Introduction Immune system is very complex system of cells and tissues with complicated interactions in order to recognize own and foreign structure.

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.

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.

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 x109/l)

GranulocytesPolymorphnuclear

AgranulocytesUnlobed nucleus

Neutrophil Eosinophil Basophil Lymphocyte Monocyte 40-70% 2-7% 0-1% 20-40% 2-10%

B, T, NK cell

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 x109/l)

GranulocytesPolymorphnuclear

AgranulocytesUnlobed nucleus

Neutrophil Eosinophil Basophil Lymphocyte Monocyte 40-70% 2-7% 0-1% 20-40% 2-10%

B, T, NK cell

Differential count

-penias & -philias IE.g neutropenia or neutrophoilia, lymphopenia or

lymphocytosis, monocytosis, etc. refer to ABSOLUTE COUNTS (NUMBERS) from the

differantial lekocyte count: Neutropenia: ANC < 1,6 x 109/l Neutrophilia: ANC > 7,0 x 109/l Lymphocytosis: Ly > 4,0 x 109/l, etc. Relative -penias or -philias: relative (%) decrease or increase in differential

leukogram in the absence of change in absolute number.

(-penias) & -philias II1. 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.

Normal count of WBC in PB: : 4,0 – 10,0 x109/l

1.Marrow pool: 90% neutrophils2.Blood pool: 3% 3.Tissue pool: 7%

LeukopoiesisIn 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.

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.

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.

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. B12 or folate deficiency). More the younger cells → shift to left (infection).

Cytoplasm: contains neutrally stained granules2 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 B12 binding protein & iii. Components of enzyme system that produce

free radicals like H2O2, which kills the microbes.

iv. Substances that facilitates chemotaxis.3. Toxic granules: During infection toxic coarse

granules are seen.

Neutrophil function

1.Phagocytosis: 1st 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

Changes in neutrophil count REACTIVENeutrophilia: ↑ 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).

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 109/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 109/l: might be bening (leukemoid reaction)WBC (ANC) 106 x 109/l: cannot be bening

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

Case I48 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 x109 with clear neutrophilia revealed. No other significant laboratory findings. Repeated BC 4 days later: 14.2 x109 . Absolutely no signs of inflammation.

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?

Changes in neutrophil count REACTIVENeutrophilia: ↑ 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).

Changes in neutrophil count REACTIVENeutrophilia: ↑ 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

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 Granules1. 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)

Functions:1.Mild phagocytosis: because less mo bile than neutrophils 2.Eosinophils collect at the sitesof allergic reactions & detoxifyInflammation 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

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

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.

Functions: 1. Mild phagocytosis2. Role in allergic reaction: Basophils release histamine, bradykinin, no. of lysosomal enzymes, slow reacting substance of anaphylaxis (SRS-A) & serotonin (5HT). These 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.

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 (kidneyShaped)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

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

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.

Variation in count: Reactive monocytosis: ↑ in monocyte 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 monocyte count Causes are:- It is rare, may be seen in hypoplastic

bone marrow.

Morphologically: small & large (LGL)Functionally: T & B lymphocytes, (and NK cells)

Small lymphocytes: 7-10 µmNucleus rounded, cytoplasm: just rim is seen.

Older cells.Large lymphocytes: 10-14 µm Nucleus is big with

indentation, definite cytoplasm is seen.

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

Variation in lymphocyte countNo. of Eosinophils and Lymphocytes are the most

sensitive to various external and internal changesLymphocytosis: ↑ in absolute lymphocyte count

Physiological 1) In healthy & young children 2) In female during menstruation

Pathological: Reactive X Malignant

Lymphocytopenia: ↓ in absolute lymphocyte count Causes are:- 1) Patients on corticosteroid &

immunosuppressive therapy 2) Hypoplastic bone marrow 3) Widespread irradiation 4) Acquired Immune Deficiency syndrome (AIDS)

Variation in lymphocyte countNo. of Eosinophils and Lymphocytes are the most

sensitive to various external and internal changesLymphocytosis: ↑ in absolute lymphocyte count

Physiological 1) In healthy & young children 2) In female during menstruation

Pathological: Reactive X Malignant

Lymphocytopenia: ↓ in absolute lymphocyte count Causes are:- 1) Patients on corticosteroid &

immunosuppressive therapy 2) Hypoplastic bone marrow 3) Widespread irradiation 4) Acquired Immune Deficiency syndrome (AIDS)

Reactive lymphocytosisLymphocytes > 4 (-5) x 109/lMorfology: usually heterogenousFlow 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)

Malignant – clonal - lymphocytosisChronic 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)

CD5CD19

CD20

CD23SmIg

CD22

CD79b

„spillover lymphomas“ dif. diagnosisDifferenciální dg. CLL MCL FLHCL

Spleen + + +/- ++

Morfology (chromatin) dense Pleomorfic‘Hairy’ chromatin „cleaved“

Imunophenotype

CD5 ++ ++ - -

CD23 ++ - - -

CD79b - + + -

SIg weak strong strongstrong

CD10 - - ++ -

Molek. genetics +12; del 13q bcl1 bcl2různé

(cytogenetics) 11q-, 17p- t(11;14) t(14;18)

LymphopeniaLymphocytes < 0,8 x 109/l1.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,

Disorders in quality of leukocytePelger 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

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.

Döhle bodie and toxic granulations

Pelger-Huët anomaly

May-Hegglin anomaly

• 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

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

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

Location

Single lymphnode, region

Advanced involvement

Generalized adenopathy

Symetry

Size

Sensitivity

Consistence (texture)

Reaction of the surrounding area

Lymphadenopathy vs. pseudolymphadenopathy

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

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.

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.

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

lymph nodes that are tendertender are more likely to be due to an infectious process,

whereas painlesspainless adenopathy raises the concern of malignancy.

Lymph node consistencyconsistency

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.

The largerlarger 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

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

• 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:

• Excision of the whole lymh node strongly

preferred

• Reasonably experienced surgeon preferred

• Fine needle biopsy must be avoided