Dr Mere Kende, MBBS, MMED (Path),MAACB (AUS), MACTM

(AUS),MACRRM (AUS)

Department of Pathology

Faculty of Health Sciences & Medicine

UPNG

Definition?

How is this done? ◦ Sample Collection ◦ Smear Preparation ◦ Preanalytical /laboratory issues

How is it useful clinically? ◦ Red Cells & Hemoglobin Issues ◦ White cell count ◦ Platelet ◦ Hematocrit/PCV ◦ Red Cell indices

Refers to; Hemoglobin measurement

Quantification of red cells, total white cells, platelets, white cell differential count &

Red Cell Indices (MCV, MCHC etc)

May include Blood Film examination depending

on laboratory protocol

Full Blood Examination or Count (FBE/FBC)

Ideally in all patients presenting into hospital

Only selected cases on OPD

Fasting not necessary

Fill request form appropriately with relevant clinical information.

Must have all necessary equipment for blood collection

Explain & reassure patient, be confident!

Ensure adequate sterile technique

Veins

Capillary

Bone Marrow

Protect yourself from blood spill & Needle stick injury

Spend time looking for vein

Avoid prolonged application of tourniquet

Avoid haemolysis/clot (decreased RBC/PLT)

Only gentle mix required

Collect into anti-coagulant tube (EDTA/heparin)

Only 5mls required (do not force blood into tubes)

Analyse within 3hrs

Evenly spread thin film –allow air-dry

Forms:

1- Tri-Potassium EDTA

2- Di-Sodium EDTA

3- Di-Lithium EDTA

EDTA is also known as Versene or Sequestrene. EDTA acts by chelating / removing ionized calcium (calcium is required for blood to clot, so when it is removed blood will not clot).

Excess EDTA

causes shrinkage of RBC’s, causing falsely / erroneously reduced hematocrit (HCT), Subsequently causes:

◦ increase in MCHC

◦ decrease in MCV (MCV and MCHC are RBC indices

PLTs disintegrate, ◦ causing erroneously high PLT count,

EDTA can induce platelet aggregation and clumping, ◦ False thrombocytopenia

◦ False lecocytosis

◦ Correct by repeating PLT count/exam Blood film

Is the anticoagulant of choice

◦ coagulation and platelet function tests,

◦ ESR (erythrocyte sedimentation rate test).

Accid mucopolysaccharide,

It acts by complexing with anti-thrombin to prevent blood clotting (antithrombin is one of the natural/physiological inhibitors of blood coagulation, which is found in vivo, this will be studied later in coagulation and hemostasis modules).

Uses: ◦ LE cells ◦ Biochemistry /Hormones

Manual Cell Count

Automated Cell Count

Blood Film Examination if required

Diagnosis of Anaemia

Screen Bacterial infection

Screen Viral Infection

Screen Blood Cancer (leukaemia)

Screen Poisoning

Monitor Treatment and emergence of complications

Use correct ref Ranges

Affected by age & sex, ethnic group, pregnancy, altitude & Physiological states (exercise)

Red Cells (4000-7000 x109 /L)

Platelets (150-400 x109 /L)

White cells (4-10 x109 /L) ◦ neutrophils, (3-8 x109/L)

◦ Lymphocytes (1.5-4.5 x109 /L)

◦ Monocytes (0.2-0.8 x109 /L)

◦ Eosinophils (0.04-0.5 x109 /L)

◦ Basophils (less than 0.2 x109 /L)

Pancytopenia- All cell Count is low Microcytosis- Smaller size red cells Macrocytosis Larger size red cells Hypochromia Faintly stained red cells

Leucopenia- Low white cell count Leucocytosis – Elevated white cell counts Lymphocytosis – Elevated lymphocytes Eosinophilia- Elevated eosinophils count Neutropenia- Low neutrophil count Neutrophilia- High neutrophil count

Thrombocytosis – Elevated Platelets Count Thrombocytopenia – Low Platelet Count

RBC count- Decreased/Increased

Hemoglobin (120-160g/L)- decreased/ increased

Red Cell Indices

RBC morphology: ◦ Fragmented RBCs, Reticulocytes, erythroblasts cells

◦ abnormal shapes & sizes

Ratio RBC’s volume : WB expressed as %

Affected by RBC count & hydration status

Used to calculate indices

Low –Anaemia

High-polycythaemia/dehydration

Decreased production

Hemoglobin synthesis/Bone Marrow problem

Increased Destruction

Blood Loss/hemolysis

Relative Dehydration Absolute PHYSIOLOGICAL

HIGH ALTITUDE

ATHLETES

BODY BUILDERS

PREGNANCY

PATHOLOGICAL

Lungs Diseases ◦ COAD ◦ Sleep Apnoea

Poisoning ◦ CO intoxication

CVS disorders ◦ CHD/right-left shunt

Neurologic disease Renal disease ◦ CRF ◦ Renal artery stenosis ◦ Focal sclerosing or membranous glomerulonephritis ◦ Renal transplantation

High –polycythaemia , dehydration

Low-Anaemia

Calculated from RBC count, PCV & Hb

Includes

◦ Mean Cell Volume (80-90fl)

◦ Mean Cell Haemoglobin (27-32pg)

◦ Mean Cell Haemoglobin Concentration (310-

350g/L)

male female

Hematocrits [hct] % 47 42

RBC [10x6 / mcl] 5.4 4.8

Hemoglobin Hb [g/dL]

16 14

MCV [fl] = hct x 10 RBC [106 /mcl]

87 87

MCH [pg] = Hb x 10 RBC [106 /mcl]

29 29

MCHC (g/dL) = Hb x 100 hct

34 34

Microcytosis (Low MCV) ◦ Iron deficiency

◦ Thalassemia

◦ Lead poisoning

◦ Sideroblastic Anemia

Megaloblastic ◦ Folate & B12 deficiency (high MCV)

Hypochromic Normochromic Microcytic Normocytic

44

45

Central color spot in the area of pallor, Resembles a target. Cause: hemolytic anemias, especially sickle cell, HbC disease, and thalassemia.

47

48

Almost spherical in shape; No area of central pallor Large spherocytes (macrospherocytes) -hemolytic anemia. Small spherocytes (microspherocytes) -severe burns. Other causes: hereditary.

50

51

There are numerous fragmented RBC's seen here. Some of the irregular shapes appear as "helmet" cells. Indicates: microangiopathic hemolytic anemia (MAHA) or other cause for intravascular hemolysis.

DIC

◦ Involved in inflammatory response ‘killing zone’

Chemotaxis: attract Neutrophils to inflammatory site

Opsonization: -coding of bacteria with IgG, complement to activate Neutrophils

Phagocytosis/degranulation- ingestion and realase of chemical stored to ‘killing’ bacteria

Respiratory burst/free radical generation-activated N

Myeloperoxidase – convert Cl-, Br- and SCN- to potent acids (hypochloride)

Elastase/collagenase -digest collagen, wall off infection

Thromoxane/platelet aggregating factors/leukotrienes -vasoconstrict, isolate inflammatory response

Neutrophils – ingest bacteria & digest it (microphages)

Basophils-

◦ contain histamine and heparin. Role?

Eosinophils- ◦ attack parasites too large for attack by macrophages &

◦ produce leukotriene C4 & PAF-involved in allergic

reactions

◦ Abundant in mucosa of GIT, resp tract & lower UT

Neutrophil Eosinophil

Lymphocyte Basophil

Monocytes- ◦ Become Tissue macrophages /RES (kupfer cells),

osteclasts, microglia, multinucleated GC)

◦ Macrophages function as Neutrophils

◦ Activated by lymphokines (T-lymphocytes)

◦ M Secrete 100 different substances (PG, IL, CSF)

Neutrophilia

Eosinophilia

Lymphocytosis

Physiological ◦ Physical stress ◦ Emotional stress ◦ Pregancy

Infections ◦ Bacterial ◦ Fungal

Inflammatory conditions ◦ Connective tissue diseases ◦ Arthritis ◦ Dermatitis

Tissue necrosis eg AMI

Hemolyitic Anemia

Myeloproliferative Disorders

Drug Therapy-corticosteroids/cytokines

Leukaemia (early myeloid cells)

Decreased Production ◦ Drugs

◦ Bone Marrow Failure

Diseases ◦ Acute Leukeamia

◦ Myelodysplasia

Nutritional Deficiencies ◦ Vitamin B12 & folate

Increased destruction ◦ INFECTIONS

(1) Drugs,

(2) parasitic infections,

(3) allergic diseases,

(4) collagen vascular diseases,

(5) malignant neoplasms,

(6) hypereosinophilic syndromes

Viral infections ◦ EBV/infectious mononucleosis ◦ CMV infection ◦ HIV infection ◦ Pertusis ◦ Other viruses ◦ TB ◦ Brucellosis ◦ syphilis

Leukaemias (CLL) usually >10 000/ul

Endocrine Disorders (thyrotoxic, adrenal failure)

Absence of spleen

Bacterial infection

Early vital infection

Carcinoma

Drugs eg steroids/cytotoxics

HIV infection

Hodgkin’s Disease

Malnutrition

Leukaemia/infection/toxicity

Auer rods- leukaemia

Toxic granulation- severe infections

Hypersegmented neutrophils -megaloblasti c anaemia/severe bacterial infections

Atypical lymphocytes: viral/EBV infection

LE cells: SLE

2-4microns

Reference ranges: 150-400,000/mcL

Half-life -4 days; transfused survive 2-3 weeks

Derived from Megakaryocytes:

60-70% in circulation-rest mainly in spleen

Membranes-receptors (vWF, Gp 1b, IIa, IIIb)

Cytoplasmic granules: dense-serotonin &ADP, alpha-Clotting factors, PDGF

Healing Wound/Tissue Damage & Clot Formation

Megakaryoblast ◦ 20-50 microns

◦ Nucleus :cytoplasm (10:1)

Pomegakaryocytes ◦ 20-50microns

Megakaryocytes--- ◦ 40-100microns

Platelets (thrombocytes) ◦ 1-4microns

Hereditary/Primary/Essential (>2million/mcl)

Acquired/Secondary/Reactive ◦ Severe hemorrhage,

◦ iron & B12 deficiency,

◦ Surgery/splenectomy (transient)

◦ Malignancy (Hodgkin’s disease, polycythemia vera),

◦ Chronic inflammatory diseases (e.g., inflammatory bowel disease)

◦ Recovery from acute infection,

◦ Drugs (e.g., vincristine, epinephrine/cytotoxic/alcohol).

Hereditary vs Acquired

Bleeding Time is prolonged

Bleeding is unusual unless count < 20,000/μL;

Platelet count < 10,000/μL------associated with serious hemorrhage

Bone Marrow Failure” ◦ Drugs, irradiation/aplastic anaemia

◦ Marrow invasion (e.g., carcinoma, leukemia, fibrosis);

Sequestration due to splenomegaly;

Accelerated destruction— ◦ Drugs: thiazides, ethanol, estrogens, sulfonamides,

quinidine, quinine, methyldopa.

• Heparin-induced thrombocytopenia is seen in 5% of pts receiving >5 days of therapy

Autoimmune destruction SLE, lymphoma, HIV.

Idiopathic thrombocytopenic purpura (ITP)

DIC—platelet consumption & coagulation factor depletion [prolonged PT & aPTT)

Platelet Count (150-450,000/mcl)

Bleeding Time <6mins

1. LG Gomella. Clinician’s Pocket Reference 11th Edition

2. Harrisons Text Book 17th Edition

3. Despopoulos. Colour Atlas of Physiology 5th Edition