Introduction to Hematology and Staining W1

Angle correction:

1\. In case of Polycythemia: high Hct, angle should be lowered \n - ensure that the smear made is not to thick

\n 2. Too low Hct: Angle should be raised

Feature of a Well Made Wedge Smear:

• Smear is 2/3 or ¾ the entire slide \n • Smear is finger shaped to straight, very slightly rounded \n at the feathery edge: widest area of examination \n • Lateral edges of the smear visible \n • Smear is smooth without irregularities, holes or streaks \n • When held up in light: feathery edge should show \n rainbow appearance \n • Entire whole drop of blood is picked up and spread

Slide Labeling

• Patient Last Name, First \n • Patient Identification Number (or lab barcode) \n • Date Y/M/D \n • Type of stain (if required) \n • Your ID (use micro format- first three letters of last name, \n initial of first name eg. Ber,N)

Cover Slip Technique

– rarely used \n – used for Bone marrow aspirate smears

\n • Advantage: excellent leukocyte distribution

\n • Disadvantage: labeling, transport, staining and storage is a problem

Romanowsky Stains

\n A group of metachromatic stains, the effect is a result of \n the combining of two dyes: acid and basic. \n • Methylene blue is a basic stain \n • Eosin is an acid stain \n • pH dependent

Methylene blue

Basic stain, high pH

Eosin

Acid stain, low pH

Types of Romanowsky Stains

**Use polychromed methylene blue and eosin.**

•Leishman, \n • Wright’s \n • Giemsa \n

**Use non-polychromed methylen blue and eosin**

• Jenner \n • May-Grunwald

Eosin + Methylene Blue = thiazine eosinate complex

• The complex will not stain any color unless a buffer is \n added: 0.05M sodium phosphate (pH 6.4) and aged distilled water (pH 6.4-6.8)

\n • Methanol is added to fix the cells on the slide

Polychroming

made by oxidizing the methylene blue to remove the methyl groups until it degrades into a group of various coloured components

Oxidative agents include: sodium bicarbonate and heat, acid bichromate, borax, sodium hydroxide and potassium carbonate and heat

There are two parts to a dye:

– the chromogen is the coloured portion, \n – the auxochrome is the charged portion

@@ \n An acid dye (eosin) has a negative auxochrome and \n unites with positive cellular components (polypeptides, \n RBC cytoplasm, eosinophil granules)@@

\
^^A basic dye (methylene blue) has a positive auxochrome \n and unites with negative cellular components (nucleic \n acids, monocyte and lymphocyte cytoplasm, basophil \n granules)^^

pH between 6.4 and 6.8

is used with most Romanowsky stains.

A phosphate buffer, such as Sorensens is used to dilute the stains and for the final rinse of the film.

f the staining solution is too basic

those cells or cell parts taking up a cationic dye (ie cell nuclei, basophil \n granules) will stain well, appearing very blue, while those cells which stain at a more acid pH will appear very pale

If the stain is too acidic,

those cells or cell parts taking up a anionic dye (ie Hb, eosinophil granules) will stain well, appearing very orange or red, while those cells which \n stain at a more basic pH will appear very pale. WBC nuclei are pale blue.

Automated Slide Stainers Disadvantages:

1. Staining process has begun, no STAT slides can be
added in the batch
2. Aqueous solutions of stains are stable only after 3-6
hours

Quick Stains Disadvantages:

1. Quality of stains especially on color acceptance
2. For small laboratories and for physician’s clinic only

Problem encountered during staining:

1\. Moth eaten RBC, heavily demarcated central pallor on the RBC \n surface (punch out), crenation, refractory shiny blotches on the \n RBC: Water Artefact

What contributes to the problem:

1. Humidity in the air as you air dry the slides.
2. Water absorbed from the humid air into the alcohol based stain

Solution:

1. Drying the slide as quickly as possible.
2. Fix with pure anhydrous methanol before staining.
3. Use of 20% v/v methanol

RBC gray, WBC too dark, Eosinophil granules are gray

Cause: Stain or buffer is to alkaline \n Inadequate rinsing \n Prolonged staining \n Heparinized sample \n Blood film too thick

RBC too bright, WBC barely visible

Cause: Stain or buffer is too acidic \n Under buffering \n Over rinsing \n Insufficient staining time

Reticulocyte Stain

• Stains RNA in developing erythrocytes just after the \n nucleus has been discarded. \n • Indication of RBC production

What is the preferred angle of a spreader when making a blood film?

40° - 45 \n

How is the end of the well made blood film described?

Feathered \n

What type of stains are used for blood films?

Romanowsky

What is a Wright’s stain made up of?

Methylene blue and eosin

What does the methylene blue (basic) stain?

nucleic acids

What does the eosin (acid) stain?

HB in RBCs

What is the preferred pH for staining blood films?

6\.6

\
6\.4-6.8