HEMA_Lec_Prelims: BASIC HEMATOLOGICAL METHODS OF EXAMINATION, QUALITY ASSURANCE PROGRAM, COLLECTION OF BLOOD, AND ANTICOAGULANTS

COMPLETE BLOOD COUNT (CBC)

basic screening test and one of the most frequently ordered laboratory procedure

Complete Blood Count

gives valuable diagnostic information about the hematologic and other body system, prognosis response to treatment and recovery

Complete Blood Count

consists of a series of tests that determine number, variety, percentage concentration, and quality of blood cells

polycythemia (↑)

a condition where there is increased hemoglobin, hematocrit, and RBC.

anemia (↓)

▪ decreased hemoglobin, hematocrit, and RBC.

1. Hemoglobin

2. Hematocrit

3. RBC Count

4. WBC Count

5. Differential Count

Parameters in CBC

Blood indicies

MCV

MCH

MCHC

Red Cell Distribution Width

❖ degree of anisocytosis ({anisocytosis}- variation of the size of the RBCs)

Reticulocytes (RETIC)

❖ young form of RBCs

❖ used to assess the erythropoietic activity of bone marrows

WBC Parameter

WBC count

Differential Count

Hg Determination

- used to diagnose diseases associated with anemia (DA) and to assess polycythemia (AP)

- used to evaluate the oxygen carrying capacity (OCC) of blood

- used to monitor effectiveness of treatment (MT)

WHITE BLOOD CELL / LEUKOCYTE COUNT

• test that measures the number of WBCs in your body • detects hidden infections

dilution for WBC Count

1:20 dilution

Leukocytosis

increased WBCs

Leukopenia

decreased WBCs

Polycythemia Vera

a chronic cancer where there is a increased RBCs

Anemia

decreased RBCs

Differential Count

checks the 5 types of WBCs in a blood smear

OSMOTIC FRAGILITY TEST (OFT)

uses graduated concentrations of saline solutions to detect spherocytes

GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) ASSAY

phenotypically detects an inherited RBC enzyme deficiency causing severe episodic hemolytic anemia

QUALITY ASSURANCE PROGRAM (QA)

sum of all those activities in which the laboratory is engaged to ensure that information generated by the laboratory is correct

Standard Operating Procedure (SOP)

- well-documented procedure for every hematology test and procedure

• includes sample collection, testing, handling, reporting

• should always be reviewed and updated

Internal QC

includes daily checking of equipment, reagents, and controls for consistent results

External QC

proficiency testing being sent by reference laboratories (RL)

Pre Analytical Phase

1. Assay Selection

2. Assay Implementation

3. Px ID and preparation

4. Spx Collection

5. Specimen Handling and Transport

6. Specimen Storage

7. Monitoring Specimen Condition

Analytical Phase

- Laboratory Staff Competence

- Assay & Instrument Selection

- Validation Processes

- Internal Quality Control (IQC)

- External Quality Assessment (EQA)

Post Analytical Phase

1. Transcription & Filing

2. Laboratory & Narrative Reports

3. Communication of Critical Values

4. Turnaround Time

5. Satisfaction Metrics

6. Physician Use of Results

7. Patient Outcomes

Capillary (Skin Puncture)

Used when small blood volumes are needed or venipuncture is difficult

Common site of skin puncture for adults

3rd or 4th finger of non-dominant hand

Common site of skin puncture for infants (<1)

Lateral heel

Order of Draw for Capillary blood collection

- Blood Gas Specimens

- EDTA tubes

- Other additive tubes

- Serum tubes

Sites of Venipuncture

Median Cubital Vein

Cephalic Vein

Basilic Vein

Venipuncture methods of collection

• Syringe and needle

• Evacuated tube system (ETS)

• Butterfly (winged) infusion se

needle angle during venipuncture

degrees 15-30 degrees insertion of needle

Application of Tourniquet

Apply 3-4 inches above site, release within 1 minute

Needle length

1 to 1.5 inches; gauge 21 or 22 commonly used

Sites if venipuncture in newborn infants up to 18 months old

-External jugular vein

-Temporal vein (scalp vein)

-Superior longitudinal sinus

Sites if venipuncture in older children (18 months to 3 years old

- Femoral Vein

- Long Saphenous vein

- Popliteal vein

- Ankle vein

Sites of venipuncture, 3 y/o to adult life

- Wrist vein

- Dorsal vein of the hand

- Dorsal vein of the ankle

Two patterns of vein

H pattern

M pattern

Veins of the antecubital fossa

- Median Cubital Vein

- Cephalic Vein

- Basilic Vein

EDTA/Versene/Sequestrene

ethylenediaminetetraacetic acid

Most commonly used anticoagulant in Hematology

Mode of Action of EDTA Tube

- bind to non-ionized form of Calcium then chelates Calcium

3 forms of EDTA

- Dipotassium EDTA

- Disodium EDTA

- Tripotassium EDTA

Dipotassium EDTA

- preferred EDTA

Advantages of EDTA

1. Preserve cellular morphology when smears are prepared within 2 hours

2. Prevent platelet aggregation

3. Prevents artefact formation even on long standing

4. Cell count ,HbF, Blood grouping and electrophoresis

5. Has a few toxic effects

Disadvantages

- After 6 hrs (inc. in MCV and OFT, dec. in ESR)

- decrease in PCV

- Not suited for coagulation studies

Citrate

- commonly used in Prothrombin time (PT) and Partial Thromboplastin Time (PTT)

Mode of Action (MOA) of Citrate

Binds calcium

3.8% Na Citrate

concentration of Anticoagulant buffer in Citrate evacuated tube

Concentration of Citrate for Westergren method of ESR (Black)

1. 1 part of 3.8 % disodium/trisodium citrate

2. 4 parts blood

Concentration of Citrate for Coagulation Testing (PT, APTT/PTT Test)

1. 1 part of 3.8 % of 3.2% aqueous sol'n disodium/trisodium citrate

2. 9 parts of Blood

Advantages of Citrate as Anticoagulant

1. For blood transfusion because citrate is less toxic.

2. TRISODIUM CITRATE is used for blood coagulation and platelet function test. 3. Buffered citrate is now commonly used because it helps stabilize plasma pH. Buffered citrate is prepared by adding citric acid to Na citrate. Buffered citrate will increase stability of Factor V and VIII.

4. For investigation of clotting disorders.

5. No known disadvantage because it has specific us

Heparin

Anticoagulant commonly sued OFT and immunotyping

> had a natural coagulation factor

2 forms of heparin

Lithium Heparin

Sodium Heparin

Uses of Heparin

1. RBC Count

2. Hemoglobin

3. Hematocrit

4. ESR

5. OFT

Disadvantages of Heparin

- not suited for coagulation

- not for WBC

- not suieted for blood smear prep/

- MOST EXPENSIVE

MOA of Oxalates

prevent coagulation by binding (chelating) calcium (Ca²⁺)

3 forms of oxalate

1. Double Oxalate

2. Lithium Oxalate

3. Sodium Oxalate

- Salts of Ammonium and Potassium

Composition of Double Oxalate

RBC Swells

What happens to RBC when it comes in contact with Ammonium oxalate

RBC Shrinks

What happens to RBC when it comes in contact with Potassium Oxalate

True

T/F

Use of double oxalate

- all RBC evaluation tests, since there is no effect on RBC

Disadvantage of Double Oxalate

- not recc. for blood transfusions

- Causes agglutination or clumping of WBCs and platelets

- not recc. for blood smear prep.

Glucose-6-Phosphate Dehydrogenase (G6PD)

D is crucial for maintaining reduced glutathione in red blood cells (RBCs).

•Clinical Indications for G6PD

- episodic hemolytic anemia

- Neonata hyperbilirubinemia

- Population screening

Sickle cell solubility test

Initial Test for Sickle cell and Hemoglobinopathy Screening

• Hemoglobin Electrophoresis

• High-Performance Liquid Chromatography (HPLC)

Confirmatory Test for Sickle cell and Hemoglobinopathy Screening

Reticulocyte count

Asses the erythropoietic activity of the bone marrow

3 Types of stains in RETICULOCYTE STAIN

1. brilliant cresyl blue

2. New methylene blue

3. Pure azure blue