2. Introduction  Immune system is very complex system of cells and tissues with complicated interactions in order to recognize own and foreign structure.  It is composed mainly of blood leukocytes & tissue cells derived from the leukocytes.  These cells work in 2 ways to prevent disease:  i) by actually destroying the invading bacteria or viruses by phagocytosis and  ii) by forming antibodies & sensitized lymphocytes, one or both of which may destroy or inactivate the invader.

3. Leukocytes: mobile units of body’s immune system. Leukocytes primarily function as defense agents outside the blood, to carry out their function, leukocytes use “seek out & attack” strategy. (i.e. they go to sites of invasion or tissue damage.) Leukocytes are formed partially in the bone marrow & in the lymphoid tissue. The main reason WBCs are present in blood is to rapidly transported from their sites of production or storage to wherever they are needed: areas of serious infection & inflammation. Thus Providing rapid and potent defense.

4. Unlike erythrocytes, (which are of uniform structure, identical function, & constant no.) leukocytes vary in structure, function & no. According to staining properties of WBCs, WBCs are divided into Leukocytes in peripheral blood (WBC) (4,0 – 10,0 x10 9 /l) Granulocytes Polymorphnuclear Agranulocytes Unlobed nucleus Neutrophil Eosinophil Basophil Lymphocyte Monocyte 40-70%2-7% 0-1% 20-40% 2-10% B, T, NK cell

5. Unlike erythrocytes, (which are of uniform structure, identical function, & constant no.) leukocytes vary in structure, function & no. According to staining properties of WBCs, WBCs are divided into Leukocytes in peripheral blood (WBC) (4,0 – 10,0 x10 9 /l) Granulocytes Polymorphnuclear Agranulocytes Unlobed nucleus Neutrophil Eosinophil Basophil Lymphocyte Monocyte 40-70%2-7% 0-1% 20-40% 2-10% B, T, NK cell Differential count

6. -penias & -philias I E.g neutropenia or neutrophoilia, lymphopenia or lymphocytosis, monocytosis, etc. refer to ABSOLUTE COUNTS (NUMBERS) from the differantial lekocyte count: Neutropenia: ANC < 1,6 x 10 9 /l Neutrophilia: ANC > 7,0 x 10 9 /l Lymphocytosis: Ly > 4,0 x 10 9 /l, etc. Relative -penias or -philias: relative (%) decrease or increase in differential leukogram in the absence of change in absolute number.

7. (-penias) & -philias II 1. Reactive. Refer to changes in blood counts as reponse to various attacks, mostly inflammatory reaction. 2. Malignant (clonal, primary) refer to changes reflecting clonal (= malignant) haematology diseases (leukemia, etc.) 3. Idiopathic: etiology and pathphysiology not known, underlying reactive or malignant ciondition ruled out 4. Congenital (inherited). Rare but exist.

8. Normal count of WBC in PB: : 4,0 – 10,0 x10 9 /l 1. Marrow pool: 90% neutrophils 2. Blood pool: 3% 3. Tissue pool: 7% Leukopoiesis In bone marrow → PHSC (Pluripotential hemopoietic stem cells) differentiates → committed stem cells → CFU-GM Granulocytes & monocytes are formed only in bone marrow, Lymphocytes are produced in MB + in various lymphoid tissues (thymus, lymphnodes, splen, etc) WBCs formed in the bone marrow are stored in the marrow & lymphocytes are stored in lymphoid tissue & small no. circulate in the blood. Mucosas etc.

9. Genesis of WBCs, distinguised by morphology Row [a] represents the myelocytes showing neutrophilic, basophilic and eosinophilic from left to right. Row [b] represents the metamyelocyte cells again starting with neutrophilic on the left.

10. Life span of WBCs Granulocytes: after released from bone marrow, 4-8 hours circulate in blood & another 4-5 days in the tissues. Survive only for few hours in serious infection Monocytes: 10-20 hrs in blood. Once in tissue they get much larger size to become tissue macrophage → life span month(s) Lymphocytes: Life span for week or years depending on body’s need. They continually circulate in blood & move from blood to tissues & from tissues to blood and again blood to tissues.

11. Neutrophils:  Size:10-14 µm in diam.  Nucleus: 1. Multilobed (1-6 lobes) therefore called polymorphnuclear leucocytes. 2. Young cell have single horse shoe shaped nucleus. 3. As the cells grow older nucleus becomes multilobed. Lobes are connected with one another by chromatin threads. 4. Arneth count: More the no. of lobes, the more mature is the neutrophil. More the no. of mature cells, Arneth count shifts to right (Vit. B 12 or folate deficiency). More the younger cells → shift to left (infection).

12.  Cytoplasm: contains neutrally stained granules 2 types of granules are present 1. Primary/ lysosomal granules: less in no. containing acid hydrolases, which can digest bacteria. After a bacterium is phagocytosed by the neutrophil, primary granules release their enzymes which remain within the neutrophil & kill the bacterium, thus no harm to innocent cells. In addition, primary granules also contain powerful broad spectrum antimicrobial polypeptide defensin 2. Secondary granules: more numerous. Contain i. Lactoferin, ii. Vit B 12 binding protein & iii. Components of enzyme system that produce free radicals like H 2 O 2, which kills the microbes. iv. Substances that facilitates chemotaxis. 3. Toxic granules: During infection toxic coarse granules are seen.

13. Neutrophil function 1. Phagocytosis: 1 st line of defence, ingest & destroy a bacteria. 2. Pyrogens & inflammatory cytokines: endogenous pyrogen which is an important mediator of febrile response to bacteria + other regulation and inflam. defense reaction

14. Changes in neutrophil count REACTIVE Neutrophilia: ↑ in neutrophils. A. Physiological 1 )Exercise, 2 )After injection of epinephrine, 3 )Pregnancy, menstruation & lactation, 4 )Newborn, 5 )After meals, 6 )Mental or emotional stress. B. Pathological 1 )Acute pyogenic (pus forming) infections, 2 )Following tissue destruction, e.g. i) Burns ii) After hemorrhage, iii) myocardial infarction, iv) After surgery v) poisoning by lead, mercury, insect venom Neutropenia: ↓ in neutrophils : 1) In children, 2) Typhoid, paratyphoid fever, 3) Viral infection, 4) Malaria, 5) Aplasia of bone marrow, 6) Bone marrow supprepression (failure).

15. Leukemoid reaction, etc. Non malignant excesive increase in leukocytes (in most cases neutrophils) in reaction to mostly inflammatory noxas. WBC can increase up to 40-60 x 10 9 /l, if neutrophilia significant left shift in differential is apparent. In most cases: sepsis (diffuse peritonitis, acute cholecystitis, acute pyelonephritis, etc.) WBC (ANC) 26 x 10 9 /l: might be bening (leukemoid reaction) WBC (ANC) 106 x 10 9 /l: cannot be bening

16. Changes in neutrophil count II Malignant neutrophilia 1. Myeloprolipherative diseases (MPD): in all cases Chronic myelogenous leukemia (CML), in some cases: Primary myelofibrosis (PMF), Polycythaemia vera (PV), Essential thrombocythaemia (ET). 2. Acute myelogenous leukemia (AML): increased number of immature precursors of neutrophils – blasts and/or promyelocytes

17. Case I 48 years old man, medium position manager of large nuclear power station. Former marathon runner, still runs 10-20km on sundays, drinks 0,7 l of french red wine in the evening, smokes 20-30 cig/day, takes sedatives every other night. Does not have any physical complain. Because of sleeplesness he visited GP on monday, with no significant physical finding but in laboratory WBC 15.4 x10 9 with clear neutrophilia revealed. No other significant laboratory findings. Repeated BC 4 days later: 14.2 x10 9. Absolutely no signs of inflammation.

18. Case I cont. GP screened the patient for infection (ENT, dental, respiratory, urology) & most common malignancies incl. CT. Nothing found. The patient was seen by a haematologist, no MPD or leukemia was found, no cytogenetic and molecular finding. What will be following GP´s approach & advise?

19. Changes in neutrophil count REACTIVE Neutrophilia: ↑ in neutrophils. A. Physiological 1 )Exercise, 2 )After injection of epinephrine, 3 )Pregnancy, menstruation & lactation, 4 )Newborn, 5 )After meals, 6 )Mental or emotional stress. B. Pathological 1 )Acute pyogenic (pus forming) infections, 2 )Following tissue destruction, e.g. i) Burns ii) After hemorrhage, iii) myocardial infarction, iv) After surgery v) poisoning by lead, mercury, insect venom Neutropenia: ↓ in neutrophils : 1) In children, 2) Typhoid, paratyphoid fever, 3) Viral infection, 4) Malaria, 5) Aplasia of bone marrow, 6) Bone marrow supprepression (failure).

20. Changes in neutrophil count REACTIVE Neutrophilia: ↑ in neutrophils. A. Physiological 1 )Exercise, 2 )After injection of epinephrine, 3 )Pregnancy, menstruation & lactation, 4 )Newborn, 5 )After meals, 6 )Mental or emotional stress. B. Pathological 1 )Acute pyogenic (pus forming) infections, 2 )Following tissue destruction, e.g. i) Burns ii) After hemorrhage, iii) myocardial infarction, iv) After surgery v) poisoning by lead, mercury, insect venom Neutropenia: ↓ in neutrophils : 1) In children, 2) Typhoid, paratyphoid fever, 3) Viral infection, 4) Malaria, 5) Aplasia of bone marrow, 6) Bone marrow supprepression (failure). Tobacco SMOKING

21.  Size:10-14 µm in diam. (2%)  Nucleus: 1. Usually (85%) cells ‘bilobed’. 2. Remaining 15% cells have trilobed nucleus.  Cytoplasm: 1. Acidophilic, appears light pink in colour after staining 2. Granular  Granules 1. Coarse, stain bright brick red with acidic (eosin) dye. 2. Granules do not cover the nucleus. 3. They contain very high peroxidase content (histaminase), lysozymes & Major Basic Protein (MBP)

22.  Functions: 1. Mild phagocytosis: because less mo bile than neutrophils 2. Eosinophils collect at the sites of allergic reactions & detoxify Inflammation inducing substances by degrading the mediators (e.g. histamine, bradikinin) 3. They enter the tissues & are specially abundant in the mucosa of respiratory tracts, GIT, urinary tract, where they provide mucosal immunity 4. Eosinophils attack parasites that are too large to be engulfed by phagocytosis. Eosinophil granules release chemicals (peroxidase) which are toxic to larvae of parasites

23. Variation in count: Eosinophilia: ↑ in eosinophils Causes are:- 1) Allergic conditions e.g. bronchial asthma, hay fever, filariasis 2) Parasitic infestation, trichinosis & schistosomiasis e.g. worms (hookworm, roundworm & tapeworm), 3) Skin disease like utricaria. 4. Malignant diseases: Myeloprolipherative diseases (esp: Eosinophilic leukemia, Systemic mastocytoisis), Hypereosinophilic syndrome, etc. 5. Connective tissue diseases: SLE, etc. Eosinopenia: ↓ in eosinophils Causes are:- 1) ACTH & steroid therapy, 2) Stressful conditions, & 3) Acute pyogenic infections

24. basophils: Size:8-10 µm in diam. Nucleus: 1. irregular bilobed, often ‘S’ shaped & its boundary is not clear because of overcrowding with coarse granules. Cytoplasm: 1. Is slightly basophilic & appear blue, it is full of granules. Granules: 1. Contain heparin, histamine & 5HT.

25. Functions: 1. Mild phagocytosis 2. Role in allergic reaction: Basophils release histamine, bradykinin, no. of lysosomal enzymes, slow reacting substance of anaphylaxis (SRS-A) & serotonin (5HT). T hese substances cause local vascular & tissue reactions that cause many allergic manifestations. 3. Liberates heparin which i. Acts as anticoagulant & keeps blood in fluid state. ii. Activates the enzyme lipoprotein lipase: removes fat particles from the blood after fatty meal.

27.  Size: Largest WBC 18-20 µm.  Nucleus: 1. Is large single unlobed, eccentric in position (present on one side of the cell). 2. It is notched/ indented (kidney Shaped) 3. It has reticulated chromatin network.  Cytoplasm: 1. Is abundant, pale blue & usually clear with no granules. Granules: 1. Sometimes contain fine purple dust like granules called Azur granules

28.  Functions: 1. Role in phagocytosis: capable of phagocytosing as many as 100 bacteria, large particles such as RBCs & malarial parasites. 2. Precursor of tissue macrophages: The mature monocyte stay in blood only for few hours & then they leave blood and enter extravascular tissue, to become tissue macrophages. 3. Role in tumor immunity: kill tumor cells after sensitization by lymphocytes

29. 4. Synthesis of: Complement & sis of biological substances: PGE & clot promoting factors. Also: Interleukin-1, Hemopoietic factors (G-CSF, GM-CSF),TNF- α, Binding proteins like transferrin, lysosomes, Proteases, Acid hydrolases 5. Key role in the lymphocyte – mediated immunity as ANTIGEN PRESENTING CELLS.

30. Variation in count: Reactive monocytosis: ↑ in m onocyte count Causes are:- 1) Certain bacterial infections, e.g. tuberculosis, syphilis & subacute bacterial endocarditis 2) Viral infections 3) Protozoal & rickettsial infections, e.g. malaria, kala azar Malignant monocytosis : Monoblasts in Acute leukemia (monocytic, myelomonocytic) Monocytes in CMML (Chronic myelomonocytic leukemia) Monocytopenia: ↓ in m onocyte count Causes are:- It is rare, may be seen in hypoplastic bone marrow.

31. Morphologically: small & large (LGL) Functionally: T & B lymphocytes, (and NK cells)  Small lymphocytes: 7-10 µm Nucleus rounded, cytoplasm: just rim is seen. Older cells.  Large lymphocytes: 10-14 µm Nucleus is big with indentation, definite cytoplasm is seen.

32. B lymphocyte: originate from BM(primary lymphoid organ), mature also in secondary lymfoid organs: lymphnode, spleen, MALT etc. 1. Involved in the humoral immunity: Antibody production, antigen presentation. 2. Flow cytometry: sIg, cytIg, CD19, CD20 T lymphocyte: processed in thymus, concerned with the cellular immunity: defensive role against viral & bacterial infections and tumor cells Flow cytometry: CD3 (compl. TCR = T cell receptor) CD4: T helpers, function: Th0, Th1, Th2 CD8: cytotoxic T cells NK cells: direct cytotoxicity: CD56, CD57

33. Variation in lymphocyte count No. of Eosinophils and Lymphocytes are the most sensitive to various external and internal changes Lymphocytosis: ↑ in absolute lympho cyte count Physiological 1) In healthy & young children 2) In female during menstruation Pathological: Reactive X Malignant Lymphocytopenia: ↓ in absolute lymphocy te count Causes are:- 1) Patients on corticosteroid & immunosuppressive therapy 2) Hypoplastic bone marrow 3) Widespread irradiation 4) Acquired Immune Deficiency syndrome (AIDS)

34. Variation in lymphocyte count No. of Eosinophils and Lymphocytes are the most sensitive to various external and internal changes Lymphocytosis: ↑ in absolute lympho cyte count Physiological 1) In healthy & young children 2) In female during menstruation Pathological: Reactive X Malignant Lymphocytopenia: ↓ in absolute lymphocy te count Causes are:- 1) Patients on corticosteroid & immunosuppressive therapy 2) Hypoplastic bone marrow 3) Widespread irradiation 4) Acquired Immune Deficiency syndrome (AIDS)

35. Reactive lymphocytosis Lymphocytes > 4 (-5) x 10 9 /l Morfology: usually heterogenous Flow cytometry: polyclonal Cuases: - infections: EBV, CMV, toxoplazmóza, HSV, measels (rubeola), Hepatitis A?B?C, HIV. Bakt: TBC, brucelosis, ricketsioss, Pertussis - Hypothyreosis, thyreoditis, Addison´s, stress - Allergy, Celiac d. Chronic inflam. Intestinal disease - Hyposplenism, asplenism, - Other: Chronic granular T lymphocytosis (LGL sy)

36. Malignant – clonal - lymphocytosis Chronic lymphocytic leukemia (CLL) and Prolymhocytic leukemia (PLL) Leukemised („spillover“) non-Hodgin lymphoma, usually indolent NHL: Follicular lymphoma, Marginal zone lymphoma (SLVL: splenic lymphoma with villous lymphocytes), Immunocytoma (Waldenström´s diasese). Rarely: Hairy cell leukemia (HCL) Aggressive NHL: mantle cell lymphoma (MCL) PTCL, Sézary´s Rarely: DLBCL (dif. Large B cell lymphoma)

38. CD5 CD19 CD20 CD23 SmIg CD22 CD79b

40. „spillover lymphomas“ dif. diagnosis Differenciální dg.CLLMCLFLHCL Spleen+++/-++ Morfology (chromatin)dense Pleomorfic‘Hairy’ chromatin„cleaved“ Imunophenotype CD5++++-- CD23++--- CD79b-++- SIgweakstrongstrongstrong CD10 - - ++ - Molek. genetics+12; del 13q bcl1bcl2různé (cytogenetics) 11q-, 17p- t(11;14) t(14;18)

41. Lymphopenia Lymphocytes < 0,8 x 10 9 /l 1. Combined immune defficiency, Wiskott-Aldrich´s, Ataxia teleangiectatica, Severe aplastic anemai, end stage of cancer, Hodgin´s lymphoma 2. Radiotherapy, chemotherapy, corticosteroid th + other immunosuppressive th, antithymocyte gl. th, Stress, Heart failure, Cushing´s 3. Intestinal lymphodrenage, drenage of Ductus thoracicus, Wiple´s, right heart failure etc. 4. Sarcoidosis, mysthenia gravis, SLE, milliar TBC, renal failure,

42. Disorders in quality of leukocyte Pelger Huët´s abnormal morphology: autosomal dominant inher. disease: hyposegmentation of the nucleus. versus Pseudopelger: severe infection, leukemias, metastatic carcinoma in bone marrow, sulfonamides May-Hegglin´s abnormality: inherited together leukopenia + Döhle´s inclusions in neutrophils + thrombocytopenia with giant platelets. Acquired: burns, infections, trauma, malignancies

43. Disorders in quality of leukocyte II Leukocyte adhesion defect (LAD): autos. Recessive membrane defect due to lack of CD18 molecule. Defect in chemotaxis, aggregability and phagocytosis. Clinicaly: necrotic skin infections, pneumonits, otitis etc. Therapy: aloSCT. Chediak-Higashi´s syndrome: autom. recessive disease with impaired migration and degranulaction of granulocytes. Comes with albinism, nystagmus, photophobia, mental retardation, frequent bacterial infections, peripheral neuropathy. Therapy: aloSCT etc.

44. Döhle bodie and toxic granulations

45. Pelger-Huët anomaly

46. May-Hegglin anomaly

48. General consideration As many as 56% seemingly healthy adults have palpable lymph nodes Further follow up is necessary: In individuals < 30 years: 80% benign etiology In individuals > 50: 60% malignant etiology

49. Adenopathy, general approach Further investigation of respective adenopathy must be driven by clinical judgement and by proper assesment of all circumstances Possible cause Confirmed cause History Physical exam. Lab. exam. histology

50. Assessing adenopathy: History Age Tumor or TBC in the history Allergy Drugs/medication Duration of adenopathy Associated complains (symptoms): fever, night swetting, malaise, weight loss, artralgy, sore throat, fatigue etc. Smoking Travels

51. Assessing adenopathy: Physical examination Location Single lymphnode, region Advanced involvement Generalized adenopathy Symetry Size Sensitivity Consistence (texture) Reaction of the surrounding area Lymphadenopathy vs. pseudolymphadenopathy

52. Causes of lyphadenopathy Infections: EBV (IM), CMV, IH, postvaccinal lymphadenitis, adenovirus, VZV, HIV, HTLV-I Staphylococcus, Streptococcus spec.,TB, atypical mycobacteria, syphilis, cat scratch disease, Chlamydias (lymf. venereum) Toxoplasmosis, histoplasmosis, coccidiomycosis, Scrub typhus, filariosis Autoimmune disorders: RA, SLE, dermatomyositis, MCTD, Sjögren´s

53. Causes of lyphadenopathy Allergy & hypersensitivity: Serum sickness, silicone reaction, vaccination, graft vs. host disease. Drugs: diphenylhdantoin, carbamazepin, gold, allopurinol, indomethacin, sulfonamides, hydralazines etc. Miscellaneous Benign Disorders: Hypothyreoidism, sarcoidosis, amyloidosis, dermathopathic lymphedanopathy, hypertriglyceridemia, extramedullary hematopoiesis Unusual causes of Lymphadenopathy: Kikuchi´s, Rosi-Dorfman´s, Inflammatory pseudotumor of lymphnodes, Vascular transformation of sinuses, Gaucher´s, Nieman-Pick, Letterer-Siwe´s, Wipple´s, etc.

54. Causes of lyphadenopathy Potentially malignant causes: Angiofollicular lymh node hyperplasia (Castleman´s disease) Lymphomatoid granulomatosis Wegener´s granulomatosis Malignant causes: Hematologic: Hodgkin´s, NHL, chronic lymphocytic leukemia (CLL), Waldenström´s disease, some acute leucemias (ALL), systemic mastocytosis. Rare: multilple myeloma Metastic cancers: breast, lung, renal, stomach, melanoma etc.

55. Frequent causes of adenopathy according to the location Inguinal (or axillar) adenopathy  1cm: usually benign Cervical adenopathy: infections, carcinomas (consistency), lymphomas. Sialoadenitis (pseudolymphadenopathy) Mediastinal adenopathy: lymphomas (mediastinum anterior), sarcoidosis, metastatic cancer Isolated axillar adenopathy: infection, breast Ca, lymphoma Isolated inguinal adenopathy (significant): infection (also veneral), lymphoma, metastatic Ca (consistency) Generalized adenopathy: infection (EBV, HIV, etc.), malignant lymphomas, CLL

56. tender lymph nodes that are tender are more likely to be due to an infectious process, painless whereas painless adenopathy raises the concern of malignancy. consistency Lymph node consistency lymph nodes containing metastatic carcinoma are rock hard, lymph nodes containing lymphoma are firm and rubbery, lymph nodes enlarged in response to an infectious process are soft. larger The larger the lymph node, the more likely a serious underlying cause exists, and lymph nodes greater than 3 to 4 cm in diameter in an adult are very concerning

57. Initial evaluation of adenopathy History, physical examiniation: done Lab: Full blood count (smear), ESR, serology, blood cultures, Chemistry includes: LDH, beta2 microglobulin, TSH If carcinoma suspected: tumor markers (CA 15-3, Ca 125, CEA, NSE, etc.) If sarcoidosis suspected: angiotensin-coverting enzyme Imaging studies: Chest X-ray, abdominal ultrasound CT, MRI, PET

58. Lymph node biopsy: when Constitutional symptoms (weight loss, fever, night sweats) otherwise unexplained Persistent adenopathy > 4 - 6 weeks, otherwise unexplained Increasing size of the lymph node for several weeks Appearence of additional lymph nodes Abnormal blood test results (anemia, elevetad ESR, LDH, liver chemistries), otherwise unexplained Abnormal chest radiograph (e.g. mediastinal adenopathy ) Must be done after a complex clinical judgement, but usual indications are:

59. Lymph node biopsy: how Excision of the whole lymh node strongly preferred Reasonably experienced surgeon preferred Fine needle biopsy must be avoided