INVESTIGATIONS OF BLOOD DISEAsES

Presented By: Aayushma Phuyal Roll no: 30 BDS 4th year

CONTENTSIntroductionCollection of blood sampleHemocytometryInvestigation of blood diseases Red Blood Cell disorder: RBC count RBC indices Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH) Mean corpuscular hemoglobin concentration (MCHC) Mean corpuscular diameter (MCD) Hemoglobin Hematocrit Erythrocyte sedimentation rate (ESR)

White blood cell disorder Total WBC count Differential count

Test for assessing hemostatis Platelet count Bleeding time Prothrombin time (INR) Von Willebrand’s antigen

Introduction Hematology is the study of blood. Blood

along with the cardiovascular system performs the

functions of transport, regulation of body

temperature and protection.

It is very useful in the diagnosis and

prognosis of disease

CLASSIFICATION Disorders involving RBC’s

AnemiaClassification Nutritional deficiency anemia

• Iron deficiency • Pernicious anemia• Folate deficiency anemia

Hemolytic anemia • Thalassemia • Sickle cell anemia• Erythroblastosis fetalis

Aplastic anemia Polycythemia

Disorders involving WBC’s

Agranulocytosis

Cyclic neutropenia

Lazy leukocyte syndrome

Chédiak-Higashi syndrome

Infectious mononucleosis

Leukemia

Disorders involving plateletsPurpura

Diseases involving specific blood factors

Hemophilia

Von Willebrand’s disease

Collection of blood sample

Asepsis

Condition of being free from septic or infectious

material – bacteria, viruses etc

Sterilization of equipment

Syringes, needles, lancets, cotton and gauze swabs

Sources and amount of blood sample

Blood sample

Capillary blood

Venous blood

Arterial blood

Cardiac catheterization

HEMOCYTOMETRY

Procedure of counting the number of cells in a

sample of blood, the red cells, the white cells,

and the platelets being counted separately.

Principle The sample of blood is diluted in a special pipette and

placed in a capillary space of known volume between a

specially ruled glass slide and a cover slip

The cells spread out in a single layer makes counting easy

Knowing the dilution employed, the number of cells in

undiluted blood can be easily calculated

Hemocytometer

Consists of the following

Diluting pipettes

Also called “cell pipettes” or “blood pipettes”

RBC and WBC pipettes

Counting chamber

Coverslips

RBC and WBC diluting fluids

Watch glasses, spirit swabs, blood lancet etc

Hemocytometer

The Total WBC Count

Principle

A sample of blood is diluted with a diluting fluid

which destroys the red cells and stains the nuclei

of the WBC.

The cells are then counted in a counting chamber

and their number in undiluted blood reported as

leucocytes/mm3

Normal leucocyte count: 4000 – 11,000 cells/mm3

Leucocytosis

Increase in WBC count beyond 11,000 cells/mm3

Physiological leucocytosis

Infants

Pregnancy

Physical exercise

Mental stress

Parturition

Extremes of temperature

Pathological leucocytosis

Acute infection with pyogenic bacteria

Myocardial infarction

Acute hemorrhage

Burns

Malignancies

Surgical operations

Leucopenia

Decrease in the number of white cells below the 4000/mm3

level

Pathological leucopenia

Infection with non pyogenic organisms

Viral infections

Drugs

Repeated exposure to X-rays and radium

Malnutrition

Hypoplasia and aplasia

Hemopoetic disorders

Differential leukocyte count

• Leishman stain• Wright stain• Giemsa• May Grunwald Giemsa stain

Principle

A blood film is stained with Leishman’s stain and scanned

under oil immersion.

The different types of leukocytes are identified and their

percentage distribution is calculated.

Ideal film thickness

Thin blood film Thick blood film

Agranulocytes

Granulocytes

WBC count

Increased Decreased

Leukemia

Infectious mononucleosis

Leukemoid reaction

Agranulocytosis

Cyclic neutropenia

( < 2000 cells/ mm3 )

Rhythmic3 week cycles

Normal blood count (4-5 days)↓

Decreased neutrophil count (increase in mono & lymphocytes; peak – completely disappear)

↓Cells reappear in 4-5 days

↓Normal blood count

absolute neutrophil count

remaining above 30,000/mm3

Acute leukemia

WBC > 1,00,000/cc

Increased BT and CT

Chronic leukemia

WBC > 5,00,000/cc

Anemia

Thrombocytopenia

Myeloblast/ undifferentiated myelocyte

Stem cell leukemia - undifferentiated

Pictures Of Blood

Normal human blood

White Cell Red Cell

Platelet

Blood with leukemia

BlastsRed Cell

Platelet

White Cell

WBC count

Qualitative defects Quantitative defects

Lazy leucocyte syndrome

Chédiak Higashi Syndrome

Agranulocytosis

Cyclic neutropenia

Laboratory findings

Lymphocytosis (Increased WBC’s)

Atypical lymphocytes (horseshoe shaped or

indented nucleus, foamy cytoplasm)

Antibodies to EBV

Paul-Bunnell test

Monospot test

Infectious mononucleosis

Paul Bunnel test (serological test)

Heterophile test

Ig M antibodies elicited by EBV infection

Acute phase – 80 -90% exhibit these Abs

Property - Agglutinate sheep erythrocytes

Titre of agglutinins & hemolysins against sheep RBC increased

4th week – titer decreases 3 month – disappears

Paul Bunnel test (serological test)

Serum + equal volume of 1% sheep erythrocyte suspensionIncubated at 370 - 4 hrs

Examined for agglutination

Infectious mononucleosis – > 100 increase in the titre Normal serum – 1:8

1:4096

Monospot test

Commercially available

Rapid qualitative slide agglutination test

Screening tool

Agglutination of horse RBCs on exposure to EBV heterophile

AB

Tests for Hemostasis

It is a homeostatic mechanism to prevent loss of

blood.

Hemostasis involves the following steps

1. Vaosconstriction

2. Platelet plug formation

3. Formation of blood clot

4. Fibrinolysis

Bleeding time

It is the interval between skin puncture and

spontaneous unassisted stoppage of bleeding

It is an in vitro test of platelet function

• DUKE METHOD (1-5 min)

• IVY METHOD (9 min)

• SIMPLATE METHOD (>7 min)

Normal bleeding time : 1- 5 mins

Advantages Simple and quite reliable

Duke bleeding time

Ivy bleeding time

Method is more reliable than Duke’s methodNormal bleeding time with this method: 9 mins

Thrombocytopenia

1. Decreased production

Bone marrow injury or failure or depression

Bone marrow invasion

2. Increased destruction

Drugs

Immune thrombocytopenic purpura

Sequestration in spleen

Clotting time

Capillary blood clotting time:

Also called Wright’s capillary glass tube method

Results:Normal clotting time: 3 – 6 mins

Drop method

Less accurate than the Wright’s capillary glass tube method.

• Normal clotting time : 2 – 4 mins

Venous blood clotting time Also called Lee and White test tube methoda. Single test tube method b. Most widely used method c. Normal clotting time : 5 – 10 mins

Increase in CT•Hereditary coagulation disorders•Haemophilias•von Willebrand disease•Afibrinogenemia and dysfibrinogenemia•Deficiency of factor XIII •Acquired coagulation disorders•Vit K deficiency•Liver diseases•Intravascular clotting•Anticoagulant therapy•Newborns

Decrease in CT•Physiological conditions•Malnutrition•Parturition

Disease BT/CT Reason Thrombocytopenic Purpura BT-Prolonged Abnormal reduction in circulating

platelets

Familial Thrombasthenia BT Prolonged Qualitative defect in plateletPlatelet count is normal

Thrombocytopathic Purpura BT Prolonged Qualitative defect in plateletPlatelet count is normal

Thrombocythemia BT Prolonged Great increase in the platelet count – interferes with formation of thromboplastin

Abnormal aggregating agents

Von Willebrand’s Disease CT – ProlongedBT – Prolonged

Abnormality of von Willebrand factor

Parahemophilia CT – Prolonged BT – Normal

Reduction in plasma Proaccelerin (factor V)

Afibrinogenemia and Hypofibrinogenemia

CT – Prolonged BT – Normal

Little or no fibrinogen present in tissues or plasma

INR = PT test PT standard

•ISI: international sensitivity index (analytical system)•Normal value = 1 Patients on anticoagulants: 2-3

Measures extrinsic pathway activity•Prolonged Prothrombin time may be due to:• Oral anticoagulants.• Deficiency of factor I, II, V, VII or X•High levels of heparin

Activated partial thromboplastin time (aPTT)•Measures the intrinsic pathway of coagulation•Prolonged APTT •Deficiency of factors I, II, V, VIII, IX, X, XI, or XII•Heparin therapy, inhibitors to specific coagulation factors•High levels of FDPs (fibrinogen degradation products)

ISI

International Normalised Ratio (INR)

Prothrombin Time

SUMMARYBleeding Time: Duke’s method=1-5 min

Ivy method=9 min

Simplate method =>7 min

Clotting Time: Wright’s method=3-6 min

Drop method= 2-4 min

Duke’s method= 1-5 min

Lee & White method=5-10 min

The average value of BT &CT is taken into consideration while doing any

investigations of the blood diseases, any fluctuation in these values

may alter the treatment plan.

Hence these values are important to rule out various

disorders and it’s needful treatment.

• Routine blood investigations are an inherent part of pathology.

• These investigations many-a-times may be diagnostic and/or

adjunct to diagnosis for various pathologies.

• Both the peripheral blood smear and hemoglobin concentration

gives us important diagnostic clues, so understanding and

reading these basic hematological techniques will helps us to

provide a better diagnosis.

CONCLUSION

REFERENCES

ESSENTIAL OF PHYSIOLOGY BY: ABS MAHAPATRA(3rd edition)BURKET’S ORAL MEDICINE BY: GREENBERG GLICK SHIP (11TH EDITION)

SHAFER’S ORAL PATHOLOGY: (7TH EDITION)