Clinical Pathology

1st Practical Reviewer

Methods of Obtaining Blood

1. Prick Method2. Venipuncture

Prick Method

• Edge of the lobe of ear• Tip of ring finger• Plantar surface of great toe (infants)

3 mm

Prick Method

• 1st drop discarded = Tissue Factor• Blood not expressed out = Dilution w/ tissue fluid

3 mm

Venipuncture

Tourniquet Cotton Syringe

Vacutainers

Venipuncture site = Median Cubital Vein

Venipuncture

Venipuncture

YellowLight Blue

RedGreen

LavenderGray

(SPS)(Na Citrate)

(CBC)(Na Fluoride)

Blood

• Quantity = 8% of Body weight = 1/13 of total body weight = 5 – 6 L (estimated at 75cc/kg)

• Color– Arterial = Bright red (due to oxyhemoglobin)– Venous = purplish red (reduced Hb)– Coal gas poisoning = bright cherry red (carbon

monoxide-Hb)– K chlorate poisoning = chocolate red (metHb)

Blood

• Reaction = pH 7.4-7.45 (alkaline)

• Sp.gravity = 1.045-1.075

• Viscosity = 5-6x that of water

Complete Blood Count (CBC)1. Hb2. Hct3. WBC count4. RBC count5. Differential Count

Hemoglobin

• Sahli’s Method• Cyanmethemoglobin

Hemoglobin – SAHLI’s Method

COMPARATOR BLOCK - for comparison

Hemoglobin – SAHLI’s Method

• Principle: – Hb (red) Acid Hematin (brown) – by addt’n of 0.1 N HCl

Sahli’s Hemoglobinometer• Comparator block•Graduated tube (with 0.1 N HCl)•Pipette (20 cu.mm)

Hemoglobin – SAHLI’s Method

Hemoglobin – SAHLI’s Method

• NV:– Conventional: 12.0-16.0 g/dl– S.I.: 120-160 g/L

example:yellow calibration reading is 14.0 therefore:14.0 x 10 = 140 grams/L

Hemoglobin – CYANMETHEMOGLOBIN

• Principle: – OxyHb MetHb by Ferricyanide– MetHb CyanmetHb by Cyanide

end product yields Amber colored solution to be read on the spectro

calibrated at 540 nm.

Hematocrit (or Packed Cell Volume)

• Adam’s Microhematocrit

Definition: Hct = ratio of the volume of RBC to that of the whole blood

Hematocrit (PCV) – ADAM’s MICROHct

Heparinized capillary tube

Hematocrit (PCV) – ADAM’s MICROHct

plain capillary tube with blood (sealed at one end)

centrifuged capillary showing separation of plasma, buffy coat and RBC layer

Hematocrit (PCV) – ADAM’s MICROHct

Microhematocrit Graphic Reader

Hematocrit (PCV) – ADAM’s MICROHct

NV:•Adult males 47 vol % or 0.47•Females 42 vol % or 0.42•At birth 56 vol % or 0.56

Hemocytometer or Counting ChamberFor direct cell count

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Hemocytometer or Counting Chamber

Identify:

W2

Identify:

R1

Identify:

R5

Identify:

center

WBC Count

• Principle:– blood is diluted with a fluid that lyses the non-

nucleated RBC and not the nucleated WBC

Uses oxalated blood

WBC Count

• Diluting Fluid: Turck’s – Glacial acetic acid 1% with tinge of 1% Gentian violet

WBC Count

WBC Count

• no. of squares counted = 4• depth of counting chamber = 1/10 or 0.1mm• dilution = 1/20

SHORTCUT FACTOR: 50 • Normal values

S.I. 4.5 – 10.0 x 109 / L (5,000-10,000 /mm3)

WBC Count

Note: if nucleated RBCs are present, the leukocyte count can be corrected by using the following formula

• Corrected WBC = Total WBC count x 100100 + No. of nucleated

RBC

Differential Leukocyte Count

A. Preparation of Blood Smears

Differential Leukocyte Count

• Purpose:– to establish the percentage distribution of the

different leukocytes

Differential Leukocyte Count

B. Staining Procedures– Giemsa Stain– Wright’s-Giemsa Stain– Rapi-Stain• Solution A = Methanol• Solution B = Methylene Blue• Solution C = Eosin

Differential Leukocyte Count

C. Diff Count

microscope

Stained slide

Tally counter

Differential Leukocyte Count

(unsuitable) erythrocytes are scattered but their three-dimensional structure is difficult to observe

(suitable) erythrocytes are uniformly distributed and their three-dimensional structure is well observed (the central part is bright)

(unsuitable) erythrocytes are stacking

Differential Leukocyte Count

• Pathway for Diff Count

Differential Leukocyte Count

Differential Leukocyte Count

• NV:% SI

Myelocytes 0%

Juvenile 0 - 1% 0 - 0.01

Stabs 0 – 5 % 0 – 0.05

Segmenters 50 – 70% 0.50 – 0.70

Lymphocytes 20 – 40 % 0.20 – 0.40

Monocytes 0 – 7% 0 – 0.07

Eosinophils 0 – 5% 0 – 0.05

Basophils 0 – 1% 0 – 0.01

Differential Leukocyte Count

Note: if nucleated RBCs are present, the leukocyte count can be corrected by using the following formula

• Corrected WBC = Total WBC count x 100100 + No. of nucleated

RBC

Identify

Identify

Identify

Identify

Identify

Identify

RBC Count

• Principle:– Blood is diluted with a fluid that is isotonic with the erythrocytes.

Diluting fluids used for erythrocyte count do not destroy the leukocytes. These are normally so few that they do not interfere with the enumeration of the erythrocytes.

Uses oxalated blood

RBC pipet with mixture of blood and diluting fluid drawn up to 101 mark

Gower’s diluting fluid

RBC Count

• Diluting Fluid: Gower’s– Sodium sulfate, glacial acetic acid, distilled water

RBC Count

RBC Count

RBC Count

• no. of squares counted = 5/25 or 1/5• depth of counting chamber = 1/10 or 0.1mm• dilution = 1/200

SHORTCUT FACTOR: 10,000 • Normal values

S.I. 4.5 – 6.0 x 1012 / L (4,500,000-6,000,000 /mm3)

RBC Morphology

• Stained smear• Normal RBC size = 6-8 um• Diameter = ½ of

neutrophil• Central pallormicroscope

Stained slide

RBC Morphology

RBC Morphology

RBC Morphology

Red Cell Indices

• MCV = micro/macrocytosis• MCH = hypo/hyperchromic• MCHC = spherocytosis

*Review page 12 of manual =)*Study formulas, normal values, and interpretations

Reticulocyte Count

• New Methylene Blue Method– Procedure A– Procedure B

• Unopette Method

Reticulocyte Count - New Methylene Blue Method (Procedure B)

Stained slide

Reticulocyte Count - New Methylene Blue Method (Procedure B)

Reticulocyte Count - New Methylene Blue Method (Procedure B)

Reticulocyte Count - New Methylene Blue Method (Procedure B)

Reticulocyte Count - New Methylene Blue Method (Procedure B)

Reticulocyte Count - New Methylene Blue Method (Procedure B)

Reticulocyte Count in % = No. of reticulocytes counted x 1001,000

NV:S.I.: 5-15 x 10-3 (0.05-1.5%)

Reticulocyte Count – Unopette Method

Reticulocyte Count – Unopette Method

Bleeding Time

• Ivy Method• Duke Method

Measures vascular integrity and platelet function in response to injury

Bleeding Time – IVY METHOD

• Principle:– BP cuff is placed on the upper arm and maintained at 40 mmHg– A standardized incision is made on the volar surface of the

forearm and the blood is blotted with filter paper every 30 seconds until it stops flowing

Bleeding Time – DUKE’s METHOD

• Principle:– Prick the patient and the blood is blotted with filter

paper every 30 seconds until it stops flowing

Clotting Time

• Lee and White• Capillary Glass Tube Method• Slide Method

Clotting Time – WHOLE BLOOD CLOTTING TIME (LEE and WHITE)

• Principle:– Measure the intrinsic coagulation mechanism– Venous coagulation time is the length of time required for

a measured amount of blood to form a clot in vitro under standard conditions

– Dependent on the blood clotting factor necessary for thromboplastinogenesis and on the amount of available fibrinogen

Clotting Time – WHOLE BLOOD CLOTTING TIME (LEE and WHITE)

• Tube 1 = Tissue Factor• Tube 3 = last blood withdrawn• N.V.: 5-15 minutes

Coagulation Time – PT/PTT

• PT = Extrinsic Factor (thromboplastin)• PTT = Intrinsic Factor (activator –kaolin)

Clotting Time – CAPILLARY GLASS TUBE METHOD

Clotting Time – SLIDE METHOD

Clot Retraction Time

• Castor Oil Method (Hirschboek Test)• Whole Blood Test Tube Method

Clot Retraction Time - Castor Oil Method (HIRSCHBOEK TEST)

• Principle– The normal blood clots retract and in doing so, express serum

• NV: 25-35 minutes

Castor oil in a vial

Clot Retraction Time – WHOLE BLOOD TEST TUBE TEST

• See page 18

Platelet Count

• Direct Counting Method: Rees-Ecker• Estimate Platelet Count from Peripheral Blood

Film• Platelet Count using Unopette

Platelet Count - Direct Counting Method: REES-ECKER

• see page 19

Platelet Count - Estimate Platelet Count from Peripheral Blood Film

• Relative Platelet count = No. of platelets in 10 OIF x 2,000

Pointed: Platelets

Platelet Count – UNOPETTE METHOD

• Each vial contains:– Ammonium

oxalate– Thimerosal

(inhibitor)– Sorensen’s

Phosphate Buffer pH 6.8

– Purified waterunopette

Counting chamber

Platelet Count – UNOPETTE METHOD

Platelet Count – UNOPETTE METHOD

• Platelet/cu.mm. = Total No. Platelets in 25 squares x 1,000

BONE MARROW

Demo Slides