Clinical Pathology

1st Practical Reviewer

Methods of Obtaining Blood

1. Prick Method 2. Venipuncture

Prick Method

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

Prick Method

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

Venipuncture

Tourniquet

Cotton

Syringe

Venipuncture site = Median Cubital Vein Vacutainers

Venipuncture

Venipuncture
(SPS)
(Na Citrate)

(CBC) (Na Fluoride)

Yellow Light Blue Red Green Lavender Gray

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. Hb 2. Hct 3. WBC count 4. RBC count 5. Differential Count

Hemoglobin
Sahli s Method Cyanmethemoglobin

Hemoglobin SAHLI s Method

COMPARATOR BLOCK - for comparison

Hemoglobin SAHLI s Method

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

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

Hemoglobin SAHLI s Method

Hemoglobin SAHLI s Method

example: yellow calibration reading is 14.0 therefore: 14.0 x 10 = 140 grams/L NV:
Conventional: 12.0-16.0 g/dl S.I.: 120-160 g/L

Hemoglobin CYANMETHEMOGLOBIN

end product yields Amber colored solution to be read on the spectro calibrated at 540 nm.

Principle:
OxyHb MetHb by Ferricyanide MetHb CyanmetHb by Cyanide

Hematocrit (or Packed Cell Volume)

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

Adam s Microhematocrit

Hematocrit (PCV) ADAM s MICROHct

Heparinized capillary tube

Hematocrit (PCV) ADAM s MICROHct

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

plain capillary tube with blood (sealed at one end)

Hematocrit (PCV) ADAM s MICROHct

Microhematocrit Graphic Reader

Hematocrit (PCV) ADAM s MICROHct

NV: Adult males Females At birth

47 vol % or 0.47 42 vol % or 0.42 56 vol % or 0.56

Hemocytometer or Counting Chamber

For 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
Uses oxalated blood

Principle:
blood is diluted with a fluid that lyses the nonnucleated RBC and not the nucleated WBC

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 100 100 + 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

Stained slide

microscope

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 threedimensional 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:
% Myelocytes Juvenile Stabs Segmenters Lymphocytes Monocytes Eosinophils Basophils 0% 0 - 1% 05% 50 70% 20 40 % 0 7% 0 5% 0 1% 0 - 0.01 0 0.05 0.50 0.70 0.20 0.40 0 0.07 0 0.05 0 0.01 SI

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 100 100 + No. of nucleated RBC

Identify

Identify

Identify

Identify

Identify

Identify

RBC Count
Uses oxalated blood

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

Gower s diluting fluid

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.

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 slide
Stained smear Normal RBC size = 6-8 um Diameter = of neutrophil Central pallor

microscope

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)

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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 100 1,000

NV: S.I.:

5-15 x 10-3 (0.05-1.5%)

Reticulocyte Count Unopette Method

Reticulocyte Count Unopette Method

Bleeding Time
Measures vascular integrity and platelet function in response to injury

Ivy Method Duke Method

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)

Castor oil in a vial

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

NV: 25-35 minutes

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 water

Counting chamber unopette

Platelet Count UNOPETTE METHOD

Platelet Count UNOPETTE METHOD

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

BONE MARROW
Demo Slides