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