Hematology

stains of blood
and bone marrow
Panoptic methods and methods for diagnostic testing
of leukemia and MDS

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

Content

Page 4

00
Warning and
Precautions

Page 5 Page 6 23 Page 24 49

01 02 03
Introduction Panoptic methods Leukemia diagnostics

Sample material Principle of leukemia detection

Fixation Sample material
Water quality for wash, rinse and dilution steps Fixation LEUCOGNOST mixture of fixatives
- Weise phosphate buffer solution
Enzyme cytochemical staining
Standard staining solutions and dry dye mixtures - LEUCOGNOST ALPA
- May-Grnwalds eosin methylene blue - LEUCOGNOST EST
- Giemsas azure eosin methylene blue - LEUCOGNOST PAS
- Wrights eosin methylene blue - LEUCOGNOST POX
- Leishmans eosin methylene blue - LEUCOGNOST AP
- LEUCOGNOST NASDCL
Fast staining method Hemacolor for microscopy - LEUCOGNOST Basic kit
Auto-Hemacolor Staining set for automatic blood
smear staining with the HEMA-TEK* slide stainer Positive and negative controls
Classification of immature-cell leukemia
Myelodysplastic syndrome
HEMATOGNOST Fe

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 Page 50 Page 51 Page 52 55 Page 56 57

04 05 06 08
Immersion/Mounting Storage Blood cell counting References

Oil immersion Storage and Blood cell counting with Literature

Mounting with Aquatex documentation - Brilliant Cresyl Blue solution and
with Entellan Brilliant Cresyl Blue ZnCl2 Certistain
and Neo-Mount - Trks solution

3
Warning and Precautions
For professional use only Protection against infection
Effective measures must be taken to protect against infection.
Contraindication
Product must be used in a manner consistent with the instructions Instructions for disposal
set forth in their respective Instructions for Use inserts and Used solutions and solutions that are past their shelf-life must be
brochures. Failure to do so may result in an increased risk of injury disposed of as special waste in accordance with local guidelines.
to the end user.
Hazard classification
Sample preparation Please observe the hazard classification on the label and the
All samples must be treated using state-of-the-art technology. information given in the safety data sheet. The product safety data
All samples must be clearly labeled. Suitable instruments must be sheet is available on the Internet and on request.
used for taking samples and for their preparation. Follow the
manufacturers instructions for application/use.

Diagnostics
Suitable controls should be conducted with each application
in order to avoid an incorrect result. Further tests must be selected
and implemented according to recognized methods. The solution
and the dye must be used by the expiry date stated.

Instructions for use

For professional use only

In order to avoid errors, the staining process must be carried out by
qualified personnel. National guidelines for work safety and quality
assurance must be followed. Microscopes equipped according to
the standard must be used.

00
Warning and
precautions

4
Introduction
Intended Use/Purpose: Indication for Use:
These microscopy dyes and chemical solution For in IVD Diagnostic use
stains are used for medical purposes, specifically
for staining cells and tissues for bacteriology or Intended User:
microbiology For professional use only

Definition of Symbols
In vitro diagnostic Authorized representative in the
Serial number
medical device European Community
Caution, consult accompanying
Catalogue number Control
documents

Batch code Positive control Contains sufficient for <rt> tests

Manufacturer Negative control Temperature limitation

Date of manufacture Sterile Upper limit of temperature

Use by (YYYY-MM-DD Sterilized using steam or

Lower limit of temperature
or YYYY-MM) dry heat
Sterilized using ethylene For IVD performance
Do not reuse
oxide evaluation only
Consult instructions Sterilized using irradia-
for use tion
Sterilized using aseptic
Biological risks
processing technique

Manufactured by: Distributed by:

Merck KGaA EMD Millipore Corporation
Frankfurter Strasse 250 290 Concord Rd.
64293 Darmstadt Billerica, MA 01821
Germany USA
+49 6151 72-0 1-978-715-4321

01
Introduction

5
Panoptic methods

02 | Content Page
Sample material 8
Fixation 8
Water quality for wash, rinse and dilution steps 9
- Weise phosphate buffer solution 9

Standard staining solutions and dry dye mixtures

- May-Grnwalds eosin methylene blue 10
- Giemsas azure eosin methylene blue 12
- Wrights eosin methylene blue 16
- Leishmans eosin methylene blue 18

Fast staining method Hemacolor for microscopy 20

Auto-Hemacolor
Staining set for automatic blood smear staining
with the HEMA-TEK* slide stainer 24

02
Panoptic methods

6
 Panoptic methods
Introduction

The combination of May-Grnwalds and Giemsas methods for blood smears is described
as panoptic staining and was developed in 1912. Nuclei are stained reddish purple,
plasma of lymphocytes and monocytes bluish, and plasma of the granulocytes light pink.
Panoptic staining can be used for sections and for staining of Spirochetes. Beside
the panoptic method, there are other standard staining methods, which will be presented
here as well.

The typical purple color of cell nuclei, is due to molecular interaction

between eosin Y and an azure B-DNA complex. Both dyes build up the complex.
The intensity of the staining depends on the azure B content and on the ratio azure
B/eosin Y. The staining result can be influenced by several factors such as the pH of
the solutions and buffer solution, buffer substances, fixation, and staining time.

The standard hematological staining solution and dye mixtures all contain eosin Y and
a mixture of methylene blue and the oxidation products. The method characteristical
composition makes the difference in the staining result. The fact is that all available
methods will visualize the sample material in a comparable style. The different methods
are suitable for all types of blood and bone marrow samples. It is more the experience or
the tradition which gives the focus to one specific method.

7
Panoptic methods
Sample material | Fixation

Sample material
Fresh and if possible native sample material should be used for the preparation of blood and bone marrow smears.
That is the starting material for all types of staining. The use of anticoagulant as EDTA should be reduced to a
minimum. Anticoagulants can reduce the stainability of blood and bone marrow samples and it could be critical
especially when the material is used for enzyme cytochemical methods. Blood and bone marrow smears must be
dried in the air for at least 30 min and fixed then with the relevant fixative and according to the instruction.
Hematological staining methods are applicable for clinical specimens in cytology as well. Specimen as urine sediment,
sputum, fine needle aspiration biopsies (FNAB), imprints, lavages can be processed with these methods in a very good
way and belong to the standard application.

Fixation
Methanol is the standard fixative for blood and bone marrow samples. Methanol is a solvent with a long tradition
as fixative in hematology. It reacts fast and is inert e.g. no changes of fine structures will be recognized when
the fixation is done with sufficient air dried material. The used methanol should have a concentration of 100 %.
A sufficient quality grade should be used to prevent problems with the stainability and the quality of the sample
material. Methanol is the gold standard as hematological fixative but it is under discussion because of the strong
hazardous classification of the solvent. Methanol has to be used in well ventilated and air-conditioned working
places. Safety protection for the staff and the working place must be organized according to the safety instructions.
The valid safety data sheet is available in the internet under: www.emdmillipore.com

Physicochemical properties of methanol Ordering information

Chemical and physical data Product Package size Cat. No.
Ignition temperature 455C (DIN 51794) Methanol 1L 1.06009.1000
Solubility in water (20C) soluble for analysis EMSURE 2.5 L 1.06009.2500
Melting point -98C ACS, ISO, Reag. Ph Eur
Molar mass 32.04 g/mol
Density (20C) 0.7902 g/cm3
pH (H2O) no data available Method
Boiling point 64.5C (1013 hPa) Fix the air dried slides for 3 min in methanol. The slide
Vapor pressure 128 hPa (20C) can be used directly after the fixation for the relevant
Explosive limits 5.5 44 % (V) staining or the slide can be stored for hours after
Flash point 10C the fixation. Material which have to be stored longer
Refractive index 1.33 should placed in a refrigerator.

02
Panoptic methods

8
 Panoptic methods
Water quality | Weise phosphate buffer solution

Water quality for wash, rinse and dilution steps

Especially hematological staining methods react very sensitive and visible of changes in the quality and pH range
of used rinsing solution. Tap water or distilled water is used normally for wash/rinse steps and for dilution of the
staining solutions. Tap water has a pH of around 7 and distilled water has a pH < 7, distilled water which is stored for
a while react more acid, the pH is lower. Acid water Eosin Y, an acid dye, reacts more intense when the used water
has an acid pH. The staining result is more reddish. Alkaline water methylene blue and the oxidation products give
a reinforcement and more intense azure metachromasy and simultaneously an extenuation of the eosin effect, the
bluish-grey shade prevails.

Weise phosphate buffer solution

The pH of the water should be in the range of 6.8 to 7.2. Optimal and reproducible staining results are accomplished
by the use of buffered solution. The phosphate buffer tablets according to Weise are especially prepared for the
buffering of water for wash, rinse and dilution steps in hematological staining methods. According to the desired
staining result, the pH of the used buffer solution is to choose. In the portfolio are offered buffer tablets according
to Weise with pH 7.2 and 6.8. We have an extra available buffer tablet according to Weise with pH 6.4 is used for
sample material where a bright orange staining of erythrocytes is required.

Preparation of buffer solution

Dissolve 1 buffer tablet in 1 L distilled water. The tablets
are very solid pressed, to keep the content and the buffer
strength at the end stable. It take some time to dissolve
the tablet in the distilled water. The buffer solution is
stable for 4 weeks and should be replaced by a fresh
buffer solution after that time.

Ordering information
Product Package size Cat. No.
Buffer tablets acc. to 1 pack 1.09468.0100
Weise pH 7.2 (100 tablets)
Buffer tablets acc. to 1 pack 1.11374.0100
Weise pH 6.8 (100 tablets)
Buffer tablets acc. to 1 pack 1.11373.0100
Weise pH 6.4 (100 tablets)

9
Panoptic methods
Standard staining solutions and dry dye mixtures
for differential blood and bone marrow smears

May-Grnwalds eosin methylene blue solution

Preparation Procedure
1. Buffer solution Air-dried smears, fixed in methanol
Dissolve 1 buffer tablet in 1 L distilled water.
Staining rack
2. Dilute May-Grnwalds solution for manual Reagents Time
staining May-Grnwalds solution 3 min
Dilute 30 mL May-Grnwalds eosin methylene blue Buffer solution (1 mL) add, mix, stain 6 min
solution with 150 mL distilled water and add 20 mL Rinse with buffer solution 1 min
buffer solution. Dry

3. Dilute May-Grnwalds solution for staining with Staining jar

staining automat Reagents Time
Slowly add 30 mL buffer solution and 220 mL distilled May-Grnwalds solution 3 min
water to 50 mL May-Grnwalds eosin methylene blue Dilute May-Grnwalds solution 6 min
solution, mix and leave to stand for 10 min. Rinse with buffer solution 2 x 1 min
Dry
4. May-Grnwalds eosin methylene blue solution
Dissolve 0.25 g May-Grnwalds eosin methylene blue Staining with staining automat
in 100 mL methanol while warming gently on a water Reagents Time
bath at 60C. Stir for 1 h, leave to stand for 24 h and May-Grnwalds solution 3 min
filter. Dilute May-Grnwalds solution 6 min
Buffer solution 1 min
5. Dilute Giemsas solution for manual staining Running water (rinse) 2 min
Dilute 10 mL Giemsas azure eosin methylene blue Dry 3 min
solution with 190 mL buffer solution, mix well, leave
to stand for 10 min, and filter if necessary.

Blood smear, May-Grnwald's stain Blood smear, May-Grnwald's stain

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Panoptic methods

10
 Panoptic methods
Staining May-Grnwalds eosin methylene blue

Blood smear, Pappenheim's stain pH 6.8 Blood smear, Pappenheim's stain pH 7.2

Pappenheims staining: Staining with May-Grnwalds Results

solution and Giemsas solution Cell type May-Grnwalds Pappenheims
Nuclei red to violet purple to violet
Staining of blood and bone marrow smears and clinical- Lymphocytes plasma blue plasma blue
cytological specimens Monocytes plasma dove-blue plasma dove-blue
Neutrophilic granules light granules light
Staining rack granulocytes violet violet
Reagents Time Eosinophilic granules granules
Cover the smear with 3 min granulocytes brick-red to brick-red to
1 mL May-Grnwald's-solution red-brown red-brown
Add 1 mL buffer solution, mix and stain 3 5 min Basophilic granules dark granules dark
Cover with dilute Giemsas solution, stain 15 20 min granulocytes violet to black violet to black
Rinse with buffer solution 1 min Thrombocytes violet violet
Dry Erythrocytes reddish reddish

Staining jar
Reagents Time Ordering information
May-Grnwalds solution 3 5 min Product Package size Cat. No.
Dilute Giemsas solution 15 20 min May-Grnwalds 100 mL 1.01424.0100
Rinse with buffer solution 2 x 1 min eosin methylene blue 500 mL 1.01424.0500
Dry solution 1L 1.01424.1000
2.5 L 1.01424.2500
May-Grnwalds eosin 25 g 1.01352.0025
methylene blue 100 g 1.01352.0100
Giemsas azure eosin 100 mL 1.09204.0100
methylene blue 500 mL 1.09204.0500
solution 1L 1.09204.1000
2.5 L 1.09204.2500

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Panoptic methods
Staining Giemsas azure eosin methylene blue

Giemsas azure eosin methylene blue solution

Staining of blood and bone marrow smears, paraffin
sections and clinical-cytological specimens

Preparation Procedure
1. Buffer solution Air-dried smears
Dissolve 1 buffer tablet in 1 L distilled water.
Staining rack/Staining jar
2. Dilute Giemsas solution for manual staining Reagents Time
Dilute 10 mL Giemsas azure eosin methylene blue Methanol 3 5 min
solution with 190 mL buffer solution, mix well, Dilute Giemsas solution 15 20 min
leave to stand for 10 min, and filter if necessary. Rinse with buffer solution 2 x 1 min
Dry
3. Dilute Giemsas solution for staining
with staining automat Staining with staining automat
Slowly add 25 mL Giemsas solution to 275 mL Reagents Time
buffer solution, mix and leave to stand for 10 min, Methanol 3 min
and filter if necessary. Dilute Giemsas solution 15 20 min
Buffer solution 1 min
4. Giemsas azure eosin methylene blue solution Running water (rinse) 2 min
Dissolve 0.76 g Giemsas azure eosin methylene blue Dry 3 min
in 50 mL glycerol and heat for 3 h at 60C on a
water bath, add 50 mL methanol, leave to stand for
5 days and filter.

Blood smear, Giemsa's stain Blood smear, Giemsa's stain

02
Panoptic methods

12
 Panoptic methods
Staining Giemsas azure eosin methylene blue

Blood smear, Giemsa's stain Blood smear, Giemsa's stain

Pappenheims staining Staining with Results with phosphate buffer acc. to Weise pH 6.8
May-Grnwalds solution and Giemsas solution Cell type Giemsas staining Pappenheims
staining
Staining rack and staining jar: see May-Grnwalds Nuclei red to violet purple to violet
solution Lymphocytes plasma blue plasma blue
Monocytes plasma dove-blue plasma dove-blue
Staining with staining automat Neutrophilic granules light granules light
Reagents Time granulocytes violet violet
May-Grnwalds solution 4 min Eosinophilic granules red to granules brick-red
Dilute Giemsas solution 20 min granulocytes gray-blue to dark violet
Rinse with buffer solution 1 min Basophilic granules dark- granules dark
Running water (rinse) 2 min granulocytes violet violet to black
Dry 3 min Thrombocytes violet violet
Erythrocytes reddish reddish
Blood nuclei bright red
parasites

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Panoptic methods
Staining Giemsas azure eosin methylene blue

Giemsa's staining of paraffin sections of bone marrow Pretreatment of bone marrow and iliac crest
or other suitable sample material biopsy materials:

Step Time Optimal results can be achieved using a mild

Deparaffinate and rehydrate the sections OSTEOSOFT decalcifying solution. To gently remove any
using standard methods calcification, the fixed biopsy materials are first placed in
Distilled water 10 sec. OSTEOSOFT for 6 hours, after which they are transferred
Undiluted, filtered Giemsa's azur eosin 15 min to histoprocessing. Smaller blocks are carefully cut and,
methylene blue solution if required, are again treated with OSTEOSOFT for an
0.1 % acetic acid 10 sec. additional 20 minutes.
Distilled water 10 sec.
2-propanol 10 sec. Results
2-propanol 10 sec. Cell type Color
2-propanol 10 sec. Cell nuclei, cells blue, dark blue
Xylene or Neo-Clear 5 min Collagen, osteoid pale blue
Xylene or Neo-Clear 5 min Eosinophilic grains red
Use Entellan new to cover the preparations moistened Acidophilic mucopolysaccharides, reddish violet
with xylene and use Neo-Mount to cover those mastocytes, cartilage matrix
moistened with Neo-Clear Acidophilic materials orange red

Notes on Giemsa's staining of paraffin sections

Always employ separate xylene or Neo-Clear rinse baths Ordering information
when Giemsa's staining paraffin sections as any ethanol Product Package size Cat. No.
traces in the solutions may result in the preparations Giemsas azure eosin 100 mL 1.09204.0100
being discolored. methylene blue 500 mL 1.09204.0500
solution 1L 1.09204.1000
2.5 L 1.09204.2500
Giemsas azure eosin 25 g 1.09203.0025
methylene blue 100 g 1.09203.0100
May-Grnwalds 100 mL 1.01424.0100
eosin methylene blue 500 mL 1.01424.0500
solution 1L 1.01424.1000
2.5 L 1.01424.2500
Methanol for analysis 1L 1.06009.1000
EMSURE ACS, ISO, 2.5 L 1.06009.2500
Reag. Ph Eur
Glycerol 85 % suitable 1L 1.04091.1000
for use as excipient 2.5 L 1.04091.2500
EMPROVE exp Ph Eur, BP

02
Panoptic methods

14
 Panoptic methods
Staining Giemsas azure eosin methylene blue

Bone marrow section, Giemsa's stain Lymph node section, Giemsa's stain

Blood smear, Pappenheim's stain Blood smear, Pappenheim's stain

Blood smear, Pappenheim's stain Blood smear, Pappenheim's stain

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Panoptic methods
Staining Wrights eosin methylene blue

Wrights eosin methylene blue solution

Staining of blood and bone marrow smears and
clinical-cytological specimens

Preparation Procedure
1. Buffer solution Air-dried smears, fixed in methanol
Dissolve 1 buffer tablet in 1 L distilled water.
Staining rack
2. Dilute Wrights solution for manual staining Reagents Time
Add 20 mL buffer solution and 150 mL distilled water Wrights solution 1 min
to 30 mL Wrights eosin methylene blue solution. Buffer solution (1 mL) add, mix, stain 4 min
Rinse with buffer solution 1 min
3. Dilute Wrights solution for staining with Dry
automated staining
Add 30 mL buffer solution and 220 mL distilled water Staining jar
to 50 mL Wrights eosin methylene blue solution. Reagents Time
Wrights solution 3 min
4. Wrights eosin methylene blue solution Dilute Wrights solution 6 min
Dissolve 0.25 g Wrights eosin methylene blue in Rinse with buffer solution 2 x 1 min
100 mL methanol, warm gently on a water bath for Dry
20 30 min or until the powder is dissolved, filter
before use. Staining with staining automat
Reagents Time
Wrights solution 3 min
Dilute Wrights solution 6 min
Buffer solution 1 min
Running water (rinse) 2 min
Dry 3 min

Result
Cell type Color
Nuclei red to violet
Lymphocytes plasma blue
Monocytes plasma gray-blue
Neutrophilic granulocytes granules light violet
Eosinophilic granulocytes granules brick-red
to red-brown
Basophilic granulocytes granules dark violet
to black
Thrombocytes violet
Erythrocytes reddish

02
Panoptic methods

16
 Panoptic methods
Staining Wrights eosin methylene blue

Blood smear, Wright's stain Blood smear, Wright's stain

Blood smear, Wright's stain FNAB (Douglas), Wright's stain

Ordering information
Product Package size Cat. No.
Wrights eosin 100 mL 1.01383.0100
methylene blue 500 mL 1.01383.0500
solution 2.5 L 1.01383.2500
Wrights eosin 25 g 1.09278.0025
methylene blue

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Panoptic methods
Staining Leishmans eosin methylene blue

Leishmans eosin methylene blue solution

Staining of blood and bone marrow smears and
clinical-cytological specimen

Preparation Procedure
1. Buffer solution Air-dried smears, fixed with methanol
Dissolve 1 buffer tablet in 1 L distilled water.
Staining rack
2. Dilute Leishmans solution for manual staining Reagents Time
Dilute 30 mL Leishmans eosin methylene blue solution Leishmans solution 1 min
with 150 mL distilled water and add 20 mL buffer Buffer solution (2 mL) add, mix, stain 5 min
solution. Rinse with buffer solution 1 min
Dry
3. Dilute Leishmans solution for staining with an
automated stainer Staining jar
Slowly add 30 mL buffer solution and 220 mL distilled Reagents Time
water to 50 mL Leishmans eosin methylene blue Leishmans solution 3 min
solution, mix and leave to stand for 10 min. Dilute Leishmans solution 6 min
Rinse with buffer solution 2 x 1 min
4. Leishmans eosin methylene blue solution Dry
Dissolve 0.12 g Leishmans eosin methylene blue in
100 mL methanol while warming gently on a water Staining with staining automat
bath at 40C, leave 5 days to mature, and filter. Reagents Time
Leishmans solution 3 min
Dilute Leishmans solution 6 min
Buffer solution 1 min
Running water (rinse) 2 min
Dry 3 min

Result
Cell type Color
Nuclei red to violet
Lymphocytes plasma blue
Monocytes plasma gray-blue
Neutrophilic granulocytes granules light violet
Eosinophilic granulocytes granules brick-red to
red-brown
Basophilic granulocytes granules dark-violet
Thrombocytes violet
Erythrocytes reddish

02
Panoptic methods

18
 Panoptic methods
Staining Leishmans eosin methylene blue

Blood smear, Leishman's stain

Blood smear, Leishman's stain

Ordering information
Product Package size Cat. No.
Leishmans eosin 500 mL 1.05387.0500
methylene blue
solution
Leishmans eosin 10 g 1.01350.0010
methylene blue

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Panoptic methods
Fast staining method Hemacolor

Hemacolor Staining set for hematological and clinical specimens

Hemacolor is a fast staining kit which allows in less Preparation
then 1 min a staining where the result is brilliant and Dissolve 1 buffer tablet in 1 L distilled water.
comparable to a Pappenheim's staining. Hemacolor
contains a fixing solution (methanol), a red (eosin Y)
and a blue (methylene blue and azure) staining solution Procedure
and buffer tablets according to Weise pH 7.2. The fact Staining in jars
that the staining solutions are separately applied has Reagents Time
the advantage that no dye precipitates occur, which are Hemacolor solution 1 5 x 1 sec.
available in standard hematological staining methods Hemacolor solution 2 3 x 1 sec.
normally. The addition of buffer tablets ensure that the Hemacolor solution 3 6 x 1 sec.
stain is very stable and highly reproducible. Buffer solution pH 7.2 2 x 10 sec.
Dry
The staining solutions are very stable in use. The buffer
solution should be replaced and refreshed after 4 weeks.
The slides must be moved in the solution because of the Staining with staining automat
short staining/reaction time it is therefore a requirement Reagents Time
to get intense and reproducible results. Hemacolor solution 1 30 sec.
Hemacolor solution 2 6 sec.
Sample material Hemacolor solution 3 4 sec.
Air-dried blood and bone marrow smears Buffer solution pH 7.2 10 sec.
Clinical specimens in cytology, e.g. urine sediment, Running water (rinse) 10 sec.
sputum, FNAB, imprints, lavages Dry 3 min

Blood smear, Hemacolor stain Blood smear, Hemacolor stain

02
Panoptic methods

20
 Panoptic methods
Fast staining method Hemacolor

Body effusion, Hemacolor stain Tumor imprint, Hemacolor stain

Result Ordering information

Cell type Color Staining sets Package size Cat. No.
Nuclei red to violet Hemacolor staining set 1 pack 1.11674.0001
Lymphocytes plasma blue Kit content:
Monocytes plasma dove-blue - Hemacolor solutions (3 x 100 mL)
Neutrophilic granulocytes granules light violet - Buffer tablets, pH 7.2 (3 tabs)
Eosinophilic granulocytes granules brick-red Hemacolor staining set 1 pack 1.11661.0001
to red-brown Kit content:
Basophilic granulocytes granules dark violet - Hemacolor solutions (3 x 500 mL)
to black - Buffer tablets, pH 7.2 (6 tabs)
Thrombocytes violet Single reagents
Erythrocytes reddish Hemacolor solution 1, 2.5 L 1.11955.2500
fixing solution
Hemacolor solution 2, 2.5 L 1.11956.2500
color reagent red
Hemacolor solution 3, 2.5 L 1.11957.2500
color reagent blue
Buffer tablets pH 7.2 1 pack 1.09468.0100
acc. to Weise (100 tablets)

21
Panoptic methods
Auto-Hemacolor

Auto-Hemacolor Staining set for automatic blood smear staining

with the HEMA-TEK* slide stainer
Procedure
* = HEMA-TEK 2000 slide Fixation, staining and rinsing of blood smears can be Place the glass slides with the material downwards to
stainer (Siemens Diagnostics) accomplished with these solutions. The methanolic dye the staining strip.
must be used. Follow the
manufacturers instructions for
solution also fixes the smears. Staining is achieved by
installation and service. the alkaline dye (azure) binding to the acidic builders of Air-dried blood smears are transported in a fully
the cells, e.g. chromatin, spongioplasm, and the acidic automatic flow system (no immersion system) over the
dye (eosin) binding to the alkaline constituents, e.g. staining strip. Pass a carefully measured, fresh quantity
cytoplasm. of dye, buffer and rinse solutions, in this order, into the
capillary space between the slides and the staining strip.
Sample material The solutions are transferred to the staining strip by
Air-dried blood and bone marrow smears. adjustable peristaltic pumps.

Preparation Technical note

The staining procedure is prepared by placing the Auto- If opened packs are not used for longer than 24 h, the
Hemacolor staining set (with folding carton) into the puncture openings of the three reagent vessels must be
automatic staining system. tightly closed to avoid evaporation, which may result in
changes in concentration and formation of precipitates.
Insert the Auto-Hemacolor staining set into the opening Remove the cannulas, and clean the cannulas and tubes
provided in the equipment. The pack must fit squarely. by placing the cannulas in methanol or ethanol and
Remove the pre-punched cardboard sections of the allowing the equipment to run. Insert the clean and dry
carton. Insert the suction cannulas into the closures and cannulas as far as the protective ring seals again, and
push them firmly through as far as the protective ring leave them until the next staining; they usually close
seals on the suction cannulas. Allow the instrument to tightly.
run in order to remove air bubbles.
An automatic sign on the equipment is given when
the amount of solution left in the equipment is only
sufficient for approx. 20 staining. The sign stain is
extinguished.

02
Panoptic methods

22
 Panoptic methods
Auto-Hemacolor

Blood smear, Auto-Hemacolor stain Blood smear, Auto-Hemacolor stain

Result
Auto-Hemacolor gives a comparable staining result to
Giemsas staining.
Cell type Color
Nuclei violet
Lymphocytes plasma blue-gray
Monocytes plasma gray-blue
Neutrophilic granulocytes granules red-violet
Eosinophilic granulocytes granules red-brown
Basophilic granulocytes granules dark-violet
Thrombocytes violet
Erythrocytes beige-brownish

Ordering information
Product Package size Cat. No.
Auto-Hemacolor 1 pack 1.15213.0001
Kit content:
- Staining solution (200 mL)
- Buffer solution, pH 7.0 (480 mL)
- Rinse solution, pH 7.0 (950 mL)

23
Leukemia diagnostics

03 | Content Page
Principle of leukemia detection 28
Sample material 28
Fixation LEUCOGNOST mixture of fixatives 29

Enzyme cytochemical staining

- LEUCOGNOST ALPA 30
- LEUCOGNOST EST 32
- LEUCOGNOST PAS 34
- LEUCOGNOST POX 36
- LEUCOGNOST AP 38
- LEUCOGNOST NASDCL 40
- LEUCOGNOST Basic kit 42

Positive and negative controls 43

Classification of immature-cell leukemia 44
Myelodysplastic syndrome 48
HEMATOGNOST Fe 49

03
Leukemia diagnostics

24
 Leukemia diagnostics
Introduction

Enzyme cytochemistry cytochemical staining serves to detect the location and

activity of cellular substances and enzyme systems. In hematology, the PAS,
peroxidase, unspecific and specific esterase and acid phosphatase reactions play a key
role in the classification of leukemia. A strongly reduced alkaline phosphatase index
is characteristic for chronic myeloid leukemia.

These fundamental chromogenic enzyme detection methods were usable in small as

well as in big labs with the use of the LEUCOGNOST staining kits. The LEUCOGNOST
staining kits assist in carrying out the semi-quantitative localization and activity
detection of enzyme systems in the cytoplasm of leukemia cells of importance
for differential diagnosis. Thus for instance the tedious weighing out of reagents on a
microbalance is fundamentally avoided. The quantities of substances in all staining
kits are such that all the cytochemical reactions can be carried out without complicated
equipment using commercially available 60 mL jars.

In addition to the LEUCOGNOST Kits with ready-to-use reagents for the determination
of alkaline leukocyte phosphatase activity as well PAS, peroxidase, specific and
unspecific esterase, and acid phosphatase reactions, all the basic reagents are available
as ready-to-use stock solutions. All the reagents are subject to stringent quality
criteria and undergo a cytochemical function test. Thought the availability of all the
items of the same source and the accompanying charts for the evaluation of the result,
it is possible to achieve a high degree of standardization in the methods.

25
Leukemia diagnostics
Principle of leukemia differentiation | Sample material

Principle of leukemia differentiation Sample material

Leukemias are autonomous tumors of the hematopoietic Only fresh, native blood and bone marrow smears should
system, mostly of the white blood cell series. be used as the starting material for all stains. The use
Leukemias are always diagnosed from blood and bone of EDTA as an anticoagulant for example significantly
marrow smears panoptically stained according to a reduces the peroxidase reaction. In cases where the
hematological standard method. While the recognition addition of an anticoagulant is required should the
of mature leukemia of the chronic lymphatic leukemia amount reduced on a minimum.
type or a chronic myeloid leukemia type is usually
unproblematic, cytological fine diagnosis within the The smears must be dried in the air for at least 30 min
group of hemoblastic immature-cell leukemia often and fixed according to the relevant instruction prior to
causes considerable difficulty. Thus errors in differential the actual cytochemical reaction.
diagnosis between acute lymphatic leukemia and acute
myeloid leukemia without certain evidence of constant
reliable morphological differential criteria such as Auer
rods, primary granulation and maturation tendency are
unavoidable.

To permit better checking of suspect differential

therapeutic effects in the treatment of acute leukemia
and to take full advantage of their use if indicated,
standardized classification of the hematopoietic
immature-cell neoplasias is essential. For over 4 decades,
stem-line specific enzyme and substrate detections
have been used with the aid of cytochemical stains in
the cytoplasms of leukemic blasts. The immunological
methods with specific fluorescence labelling of the
blastic membranes contribute to a subclassification
particularly within the group of acute lymphatic
leukemias.

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 Leukemia diagnostics
Fixative for enzyme cytochemistry

Fixative for enzyme cytochemistry

LEUCOGNOST fixing mixture is especially developed Procedure
for the fixation of blood and bone marrow smears Fix the air-dried blood and bone marrow smears in
using the various LEUCOGNOST kits. LEUCOGNOST LEUCOGNOST fixing mixture 1 3 min.
fixing mixture optimally protects enzyme activities, and
the reaction times of the different working solutions Air-dry and process immediately acc. to the protocol or
are specially matched to the fixing mixture. store at +4 to +8C until required.

Only fresh, native blood and/or bone marrow smears Note: The staining protocols and especially the reaction
should be used as the starting material for all stains. time in the protocol are associated with the fixation of
The use of e.g. EDTA as anticoagulant significantly LEUCOGNOST fixing mixture.
reduces the peroxidase reaction. In any case it is quite
unnecessary to add any anticoagulant substances.
Ordering information
The thin, air-dried blood and/or bone marrow smears Product Package size Cat. No.
should be stored for maximally 3 days prior to the LEUCOGNOST 500 mL 1.12327.0500
procedure. fixing mixture

The smears must be dried in air for at least 30

minutes and fixed in LEUCOGNOST fixing mixture
according to the relevant instructions prior to the
actual cytochemical reaction.

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Leukemia diagnostics
Staining with cytochemical reagent kit for the diagnosis of leukemia

LEUCOGNOST ALPA
Detection of the alkaline leukocyte phosphatase Preparation of staining solution*
activity in leukocytes Solution A
The determination of the activity (index) of alkaline Dissolve 4 level measuring spoonful (enclosed = 1.1 g)
leukocyte phosphatase is suitable for the cytochemical of reagent 1 in 100 mL distilled water.
differentiation of chronic myeloid leukemia from other Solution B
diseases of the myeloproliferative type, particularly from Wash the contents of one bottle of reagent 2 into the
myelofibrosis and polycythemia or other inflammatory staining cuvette with 15 mL of solution A.
or tumorous processes. Further, the index of alkaline Solution C
leukocyte phosphatase represents a simple parameter for Wash the contents of one bottle of reagent 3 into
prognosis in CML, as it reflects the different phases of a conical flask with 45 mL of solution A, shake
activity of the hematological disease. vigorously for 2 minutes and filter into the staining cell
containing solution B through a full- flow filter.
Principle Mix solutions (A + B + C) well
Alkaline leucocyte phosphatase (AP) catalyzes The reagent solution is stable for a maximum of 1 1/2
the hydrolysis of phosphate esters in alkaline solution. hours. The reagent solution is red brown and rapidly
1-naphthol released from 1-naphthyl phosphate is becomes turbid. The turbidity, however, does not
coupled to a diazonium salt to form a brown azo dye, influence the staining quality.
which is precipitated according to the locality and
the AP activity in the cell. Procedure
Steps Time
1. Fix the air-dried blood and bone 1 3 min
marrow smears in LEUCOGNOST
fixing mixture
2. Wash under running tap water 10 sec.
3. Air dry
4. Place in freshly prepared staining 10 15 min
solution*
5. Rinse with distilled water and air dry
6. Stain with Mayer's hemalum solution 5 min
7. Rinse with tap water 1 3 min
8. Air dry and cover with Aquatex and
a cover glass

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 Leukemia diagnostics
Staining LEUCOGNOST ALPA

Result
The brown reaction product is only present in the final
mature stages of granulopoiesis. Assess 100 neutrophile
ones with segmented nuclei; in the event of neutropenia
at the most up to 10 % with rod nuclei. Count according
to the degree of staining using the following 5 color
intensity steps.

Color intensity steps

0 no reaction
1 single to few granules
2 many granules localized Blood smear, LEUCOGNOST ALPA
3 granules diffuse distributed
4 cell completely overcasted with granules
5 maximum number of granules, nucleus frequently
no longer visible

Multiply the percentages determined by the factors for

the corresponding reaction classes and add the products
to obtain the dimension less ALPA index.

Normal range: 10 to 100

A reduced index is pathognomonic for the active

disease phase of chronic myeloid leukemia. Only
hemolytic anemias, iron deficiency anemias or individual Blood smear, LEUCOGNOST ALPA
virus diseases produce comparable low index values.
Normal and increased values always allow a number
of interpretations, so that they are of no significance
for differential diagnosis. Chronic myeloid leukemia in
remission can also produce normal or even increased Ordering information
ALPA values. In general the index is higher, the Product Package size Cat. No.
more extensively necrobiotic catabolic processes LEUCOGNOST ALPA 1 pack 1.16300.0002
(e.g. inflammatory tissue liquefaction) proceed in (for 12 staining batches)
inflammatory or tumorous processes. Kit content:
- Reagent 1: Tris(hydroxymethyl)-aminomethane
- Reagent 2: 1-naphthyl phosphate sodium salt
- Reagent 3: Variamin blue salt B

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Leukemia diagnostics
Staining LEUCOGNOST EST

LEUCOGNOST EST
Detection of the alpha-naphthyl acetate esterase Preparation of staining solution*
reaction in leukocytes Solution A
Esterase reactions with different substrates facilitate Dissolve 2 level measuring spoonful (enclosed, 0.8 g) of
differentiation between myeloblastic and monoblastic reagent 1 in 60 mL of distilled water.
leukemia. Apart from the naphthol AS-D chloroacetate Solution B
esterase reaction, whose reliability is comparable with Dissolve the contents of 1 bottle of reagent 2 in
that of the peroxidase reaction, the 1-naphthyl acetate 2 mL acetone, add to 60 mL of solution A and shake
esterase reaction is the most suitable for identifying vigorously for 1 minute.
monoblastic types of leukemia. Solution C
Mix 4 5 drops (0.2 mL) of reagent 3 and reagent 4,
1-Naphthyl acetate esterases accelerate the hydrolytic respectively, in an empty bottle of reagent 2 and wait
cleavage of 1-naphthyl acetate to form acetic acid and 1 minute (diazotization time).
1-naphthol, which couples with a diazonium salt to form Mix solutions B and C and filter through a full flow
a red brown azo dye which is insoluble in water. filter into the staining cell.

Note: The staining solution is stable for a maximum

of 2 1/2 hours. The staining must be conducted within
15 minutes after preparing the reagent solution. The
staining solutions must be freshly prepared immediately
before each staining process.

Procedure
Steps Time
1. Fix the air dried blood and/or 1 3 min
bone marrow smears in LEUCOGNOST
fixing mixture
2. Wash with distilled water 1 min
3. Place in freshly prepared staining 12h
solution* and incubate in the dark
4. Wash with distilled water 10 sec.
5. Stain with Mayer's hemalum solution 30 min
6. Wash under tap water 2 min
7. Air dry and cover with Aquatex and
a cover glass

The stain is stable for about 5 days without embedding

and for only a few hours when covered with immersion
oil. The stability can be extended to several months with
the use of embedding agent and a cover glass.

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 Leukemia diagnostics
Staining LEUCOGNOST EST

Result
1-naphthyl acetate esterase reacts weakly in all
hematopoietic cells. In particular monocytes, plasma
cells, erythroblasts and megakaryocytes react more
strongly. The red-brown granular color reaction in
this kit is adjusted such that practically only leukemia
monoblasts/monocytes with the highest reactivity
become stained.

To classify acute leukemia, determine the percentage of

esterase-positive blasts and taking into consideration
simultaneous differently graded peroxidase reactions, Blood smear, LEUCOGNOST EST
place in one of the categories below:

Categories
Peroxidase type below 25 % EST-pos. blasts AML,
AProL
POX-EST mixed type 25 % 50 % EST-pos. blasts
AMMoL
Esterase type over 50 % EST-pos. blasts AmoL

Blood smear, LEUCOGNOST EST

Ordering information
Product Package size Cat. No.
LEUCOGNOST EST 1 pack 1.16301.0002
(for 12 staining batches)
Kit content:
- Reagent 1: Phosphate buffer
- Reagent 2: 1-naphthyl acetate
- Reagent 3: Pararosaniline-HCI solution
- Reagent 4: Nitrite solution

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Leukemia diagnostics
Staining LEUCOGNOST PAS

LEUCOGNOST PAS
Detection of the periodic acid Schiff reaction Preparation of staining solutions*
in leukocytes Solution A
The PAS reaction is an important method for the Dissolve the contents of 1 bottle of reagent 1 in 60 mL
identification of lymphatic cell elements. Next to of distilled water and transfer to a staining cuvette.
peroxidase and esterase reactions, it is one of the three Solution B
basic cytochemical staining methods important for Dissolve the contents of 1 bottle of reagent 2 in 60 mL
differential diagnosis that are regularly carried out in of distilled water, transfer to a staining cuvette, add
acute cases of leukemia. Smears already stained by the 2 mL of reagent 3 and mix. All the reagent solutions
Pappenheim's method can additionally be stained with are colorless and stable for 3 hours.
PAS and the stains subsequently removed with 1 %
periodic acid. Procedure
Steps Time
Periodic acid cleaves neighboring carbon-carbon bonds 1. Fix the air dried blood and bone marrow 1 3 min
in polysaccharides (glycogen) when hydroxyl groups are smears in LEUCOGNOST fixing mixture
attached to both carbon atoms. The alcoholic groups are 2. Wash under running tap water 10 sec.
then oxidized to aldehydes, which can subsequently be 3. Place in solution A* 30 min
clearly revealed with Schiff's reagent (fuchsin sulfurous 4. Wash with distilled water 10 sec.
acid), producing a red stain. 5. Place in solution B* 1 min
6. Wash with distilled water 10 sec.
7. Stain in Schiff's reagent (20 to 25C, 30 min
incubate in the dark
8. Wash in distilled water 10 sec.
9. Place in solution B* 2 min
10. Place in distilled water 3 min
11. Stain with Mayer's hemalum solution 3 min
12. Wash under running tap water 3 5 min
13. Air dry and cover with Aquatex and
a cover glass

The stain is stable for about 30 days without embedding

and for only 3 days when covered with immersion oil.
The stability can be extended to several months with the
use of embedding agent and a cover glass.

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 Leukemia diagnostics
Staining LEUCOGNOST PAS

Result
All polysaccharide and in particular glycogen containing
structures are stained bright red. Blast populations that
at least partly show a characteristic coarse grained PAS
positive granulation generally belong to the lymphatic
series. Leukemia blasts of the myeloid series are
diffuse to fine grained, sometimes also coarse plaqued
and PAS positive. Normal myeloblasts, eosinophilic
ones and cells of the unaffected red blood cell series are
PAS negative by contrast. Promyelocytes, monocytes,
basophilic ones and the entire neutrophilic development
Blood smear, LEUCOGNOST PAS series demonstrate a diffuse red coloration that shows
up bright red with increasing maturity. Erythroblasts in
erythroleukemia and some extremely hyper regenerative
anaemias can demonstrate a conspicuous PAS reaction.

Blood smear, LEUCOGNOST PAS

Ordering information
Product Package size Cat. No.
LEUCOGNOST PAS 1 pack 1.16302.0002
(for 12 staining batches)
Kit content:
- Reagent 1: Periodic acid
- Reagent 2: Potassium disulfite
- Reagent 3: Hydrochloric acid
Blood smear, LEUCOGNOST PAS Schiffs reagent 500 mL 1.09033.0500
2.5 L 1.09033.2500

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Leukemia diagnostics
Staining LEUCOGNOST POX

LEUCOGNOST POX
Detection of the peroxidase reaction in leukocytes Procedure
The peroxidase reaction, specially the cytochemically Steps Time
significant myeloperoxidase reaction, is used to detect 1. Fix the air dried blood or bone marrow 1 min
myeloid cell elements, where it is possible to obtain a smears in LEUCOGNOST fixing mixture
good estimate of the degree of maturity of the maturing 2. Wash under running tap water 10 sec.
granulocytes from the intensity of the black brown color 3. Place in freshly prepared staining 10 min
reaction. solution*
4. Rinse with distilled water 10 sec.
Peroxidase are lysosomal catalases which transfer Dry in the air
hydrogen from a suitable donor (previously the 5. Stain with Mayer's hemalum solution 2 min
carcinogen benzidine, here: 4-chloro-1-naphthol) to 6. Wash with tap water 3 5 min
a peroxide (here: hydrogen peroxide). The donor 7. Air dry and cover with Aquatex and
4-chloro-1-naphthol is oxidized and converted to a cover glass
black brown insoluble dye which can be regarded as
an indicator of the peroxidase activity. The stain is stable for about 3 days without embedding
and for only a few hours when covered with immersion
Material oil. The stability can be extended to several months
Only fresh, native blood and/or bone marrow smears with the use of embedding agent like Aquatex and a
should be used as the starting material for all stains. The cover glass.
use of EDTA as an anticoagulant for example significantly
reduces the peroxidase reaction. It is in any case quite
unnecessary to add anticoagulant substances. Fine air Result
dried blood and/or bone marrow smears not more than All cells in the neutrophilic and particularly the
3 days old are required. The smears must be dried in the eosinophilic series of maturity starting with
air for at least 30 minutes and fixed according to the promyelocytes have black brown colored granules and
relevant instructions prior to the actual cytochemical are thus clearly peroxidase positive. The more mature
reaction. myeloblasts also can contain peroxidase positive
fermentation islands in their cytoplasms, even in cases
Preparation of staining solution* in which the Pappenheim's staining method shows
Dissolve the contents of 1 bottle of reagent 1 in 15 mL primary granulation at the early stage of development.
of ethanol and transfer to the staining cuvette. The great majority of normal monocytes also reacts
peroxidase positively. Their coloration is, however,
Add with stirring, 45 mL distilled water, 10 drops of significantly weaker than that of the neutrophilic and
reagent 2 and 2 drops of reagent 3. eosinophilic granulocytes. Basophilic granulocytes
and all cells of the lymphatic and erythropoietic series
Note: The reagent solution is colorless and stable are peroxidase negative.
for 3 hours.

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 Leukemia diagnostics
Staining LEUCOGNOST POX

Leukemia blast populations which react partly or

completely peroxidase-positively are evidence of acute
myeloid leukemia, as the lymphoblasts and lymphoid
cells of significance in differential diagnosis are always
peroxidase-negative. Auer bodies appear conspicuously
strongly. In the absence of a peroxidase reaction,
however, acute myeloid leukemia cannot be ruled out
from the beginning.

In order to positively identify acute myeloid leukemia

with an esterase reaction of less than 50 % positivity as
myeloblastic, promyelocytic or myelomonocytic leukemia, Blood smear, LEUCOGNOST POX
the exact percentage of peroxidase positive cells in each
blast population must be counted. Additional subdivision
of the positive cells according to degree of intensity is
not required.

In peroxidase positive leukemia, there are 3

reaction types:
POX type 1
- up to 5 % POX-positive blasts
- AML without maturation tendency;
AUL or ALL not excluded
POX type 2
- 5 % to 65 % POX-positive blasts
- AML without maturation tendency or AMMoL Blood smear, LEUCOGNOST POX
POX type 3
- over 65 % POX-positive blasts
- AML with maturation tendency up to AProL

Ordering information
Product Package size Cat. No.
LEUCOGNOST POX 1 pack 1.16303.0002
(for 12 staining batches)
Kit content:
- Reagent 1: 4-chloro-1-naphthol
- Reagent 2:
Tris(hydroxymethyl-aminomethane)-HCI buffer
- Reagent 3: Hydrogen peroxide solution

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Leukemia diagnostics
Staining LEUCOGNOST AP

LEUCOGNOST AP
Detection of the acid phosphatase reaction Procedure
in leukocytes
Acid phosphatase demonstrates specific activity in Procedure without inhibition by tartrate
almost all hematopoietic cell elements (with the Steps Time
exception of neutrophilic and eosinophilic elements) 1. Fix the air dried blood and bone 1 3 min
and this is a particularly pronounced characteristic in marrow smears in LEUCOGNOST
T-lymphoblastic cells and plasmocytoma cells. fixing mixture
2. Wash with distilled water 1 min
Acid phosphatase catalyses the hydrolysis of phosphate 3. Place in freshly prepared staining 23h
esters in an acidic medium. Under suitable conditions, solution* and incubate in the dark
naphthol AS-BI is released from naphtho-AS-OL 4. Wash with distilled water 10 sec.
phosphate and coupled with a diazonium salt to give a 5. Stain with Mayer's hemalum solution 15 min
red brown azo dye which is precipitated in the cell. 6. Wash under tap water 2 min
7. Air dry and cover with Aquatex and
a cover glass
Preparation of staining solution*
Dissolve the following in sequence in 60 mL of distilled The stain is stable for about 10 days without embedding
water: 2 mL of reagent 1 and 3 level measuring spoonful and for only a few hours when covered with immersion
(enclosed, 0.8 g) of reagent 2. oil. The stability can be extended to several months with
the use of embedding agent and a cover glass.
Mix 4 5 drops of reagent 3 and reagent 4, respectively,
in a small test tube, wait 1 minute and then add to Procedure with inhibition by tartrate
the solution. The individual reaction steps and solutions are
identical with those of the procedure without tartrate
Filter the reagent solution into the staining cuvette inhibition. Only the staining solution is slightly
through a full flow filter. modified by dissolving a further 4 level measuring
spoonful (enclosed = 0.35 g) of reagent 5.
Note: The reagent solution is stable for a maximum
of 3 1/2 hours. The staining must be conducted within
15 minutes of preparing the reagent solution.

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 Leukemia diagnostics
Staining LEUCOGNOST AP

Result
In contrast to other lymphatic cell elements,
T-lymphoblastic cells demonstrate characteristic red
brown fermentation islands. Thus with the aid of
acid phosphatase, it is in many cases possible to obtain
a clear identification of otherwise cytochemically
undifferentiable leukemia.

Addition of tartrate to the reaction mixture inhibits

the normal phosphatase activity so that little or no
coloration takes place in the blood and bone marrow
Blood smear, LEUCOGNOST AP cells. The acid phosphatase (isoenzyme 5) alone in
the characteristic cells of hair cell leukemia is tartrate
resistant in this procedure and can therefore be used
as a diagnostic characteristic.

Blood smear, LEUCOGNOST AP

Ordering information
Product Package size Cat. No.
LEUCOGNOST AP 1 pack 1.16304.0002
(for 12 staining batches)
Kit content:
- Reagent 1: Naphthol AS-OL phosphoric acid
- Reagent 2: Sodium acetate
- Reagent 3: Pararosaniline-HCI solution (2 N)
- Reagent 4: Nitrite solution 4 %
Bone marrow biopsy, LEUCOGNOST AP - Reagent 5: Di-sodium tartrate

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Leukemia diagnostics
LEUCOGNOST NASDCL

LEUCOGNOST NASDCL
For the detection of naphthol AS-D chloroacetate Procedure
esterase in leukocytes
Naphthol AS-D chloroacetate esterase brings about the Fixation
enzymatic hydrolysis of naphthol AS-D chloroacetate to a Steps Time
naphthol compound. This in turn reacts with a diazonium 1. Immerse the air-dried smear in 5 min
salt to form an insoluble red-violet dye. LEUCOGNOST fixing solution
2. Immerse in distilled water 5 min
Preparation of staining solution* 3. Air-dry and process immediately or 5 min
store at +4 to +8C until required.
Solution A
Dilute 10 mL of reagent 1 with 60 mL distilled water. Staining procedure
Add the contents of bottle 2 and rinse out the bottle Steps Time
2 3 times with a few milliliter of buffer. 1. Incubate in the freshly prepared 30 min
Solution B staining solution* at room
Add 15 drops of reagent 3 to bottle 4, mix and allow temperature
to incubate for 2 minutes. 2. Place in distilled water 5 min
Solution C 3. Counter-stain in Mayer's hemalum 5 min
Add solution B to solution A and rinse out the bottle 4. Rinse with tap water 5 min
2 3 times with a few milliliter of substrate buffer 5. Air-dry and cover with Aquatex and
mixture. cover glass
Prepare the staining solution immediately prior
to use. If the smear has not been mounted, the stain is stable
for a few days only; if immersed in oil, the stain is stable
for a few hours only. Use of a mounting agent and cover
glass prolongs stability to several months.

Result
Naphthol AS-D chloroacetate esterase reacts clearly
with both mature and immature granulocytes. Intensive
enzyme activity can also be shown in the case of
myelocytes, metamyelocytes, stab cells and mast cells.
Activity may be detected in myeloblastic leukemia
cells, promyelocytes and Auer bodies. Monocytes show
such activity only rarely. Eosinophiles, basophiles,
megakaryocytes, lymphocytes, plasma cells and red cell
precursors show no or at most very weak reaction.

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 Leukemia diagnostics
LEUCOGNOST NASDCL

Blood smear, LEUCOGNOST NASDCL Blood smear, LEUCOGNOST NASDCL

Bone marrow biopsy, LEUCOGNOST NASDCL Bone marrow biopsy, LEUCOGNOST NASDCL

Ordering information
Product Package size Cat. No.
LEUCOGNOST NASDCL 1 pack 1.16198.0001
(for 12 staining batches)
Kit content:
- Reagent 1: Tris buffer concentrate
- Reagent 2: Naphthol AS-D chloroacetate
- Reagent 3: Sodium nitrite solution
- Reagent 4: Fast Red violet LB salt solution

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Leukemia diagnostics
Staining LEUCOGNOST Basic kit

LEUCOGNOST Basic kit

The LEUCOGNOST Basic kit contains reagents that are used with the
various LEUCOGNOST kits and are specially matched in quantity to
the kits. These reagents fit perfectly to the single LEUCOGNOST kits
and allow intense and reproducible results. The LEUCOGNOST Basic kit
complete the available LEUCOGNOST kits.

Ordering information
Product Package size Cat. No.
LEUCOGNOST Basic kit 1 pack 1.16305.0001
Kit content:
- Reagent 1: Mayer's hemalum (500 mL)
- Reagent 2: LEUCOGNOST fixing mixture (2 x 500 mL)
- Reagent 3: Schiff's reagent (500 mL)
- Reagent 4: Ethanol absolute (500 mL)
- Reagent 5: Acetone (20 mL)
- Reagent 6: Aquatex (20 mL)

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 Leukemia diagnostics
Positive and negative controls

Positive and negative controls

To confirm the result in the face of possible unspecific reactions, it
is necessary to conduct a positive and negative control with each
cytochemical staining batch.

Positive control
The simplest method here is to simultaneously stain normal blood and
bone marrow smears. The cells of these smears with their typical color
reactions and color intensities serve as references. In addition, normal
cell types in the pathological preparation provide a good internal
standard.

Example: A normal blood smear is included in the peroxidase reaction.

Segmented leukocytes must give a strong positive reaction, lymphocytes
a negative reaction. If the control gives both these results, it can be
assumed that the peroxidase stain was conducted properly.

Negative control
To do this, a second smear from the patient is used. The smear is treated
in the same way as the first, except that the actual substrate is left out.

Example: A second smear from the patient is stained alongside the first
in the esterase reaction. In this parallel stain, however, the substrate,
alpha-naphthyl acetate, is left out so that no color reaction can take
place. If it still takes place, an unspecific reaction is involved which must
not be assessed as positive in the first smear either.

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Leukemia diagnostics
Classification of immature-cell leukemia

Classification of immature-cell leukemia

Acute leukemia is always diagnosed independently of the total number of cells on the basis of their blast population
in panoptically stained blood and bone marrow smears. Further attempts at the differentiation only on basis of
cytomorphological criteria are, however, not very reliable. The classification of acute leukemia is therefore heavily
based on the stem-line specific enzyme detections within the cytoplasma of leukemic blasts. However, a widely held
misunderstanding must be pointed out: cytochemical stains cannot be used as an unselected screening method for
better primary detection of acute leukemia.

Cytochemical stain of normal cells

The majority of cases of acute leukemia in adults detected from blasts can be accurately classified from a typical
pattern of findings based on PAS, Peroxidase (POX), and alpha-naphthyl acetate esterase (EST) reactions see table
Cytochemical stain of normal cells below.

Cytochemical stain of normal cells

Cell type POX PAS ALPA AP EST NASDCL
Peroxidase Periodic acid Alkaline Acid Alpha-naphthyl Naphthol AS-D
Sudan Black B Schiff reaction phosphatase phosphatase acetate esterase chloroacetate esterase
leukocyte granular ++++ fine granular +++ + to ++++ diffuse (+) (+) +
metamyelocyte granular ++++ fine granular +++ negative to + diffuse (+) negative +
myelocyte granular ++++ fine granular ++ negative to + diffuse + negative ++
promyelocyte granular ++ fine granular + negative to + diffuse + negative +
myeloblast negative to negative negative negative (+) (+)
granular +
eosinophilic eosinophil negative to + negative diffuse ++ negative negative
granules positive
basophilic granular ++++ negative to + negative negative negative negative
monocyte weak granular + negative to + negative + to ++ (+) negative
megakaryocyte negative fine granular + negative diffuse +++ ++++ negative
normoblast negative negative negative ++ ++ negative
lymphocyte negative a few fine to negative in T cells (+) focal negative
middle +
plasma cells negative negative negative diffuse ++ negative

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 Leukemia diagnostics
Classification of immature-cell leukemia

Cytochemical stain of acute leukemia

In general there is no problem in determining to the particular cytochemical subtype and therefore making a
differential diagnosis in acute leukemia on basis of the cytochemical constellation in approx. 95 % of cases
see table Cytochemical stain of acute leukemia below.

Cytochemical stain of acute leukemia

FAB-Classification POX PAS EST NASDCL AP
Peroxidase Periodic acid Alpha-naphthyl Naphthol AS-D Acid
Sudan Black Schiff reaction acetate esterase chloroacetate esterase phosphatase
M1 3%+ negative to negative negative negative
fine granular +
M2 ++ negative to negative + to ++ negative
fine granular +
M3, M3 Var +++ negative to negative + to ++ negative
fine granular +
M4, M4 Eo >5%+ negative to ++ (+) negative
fine granular +
M5a, b negative to +++ ++++ negative
fine granular +
M6 + in myeloic blasts coarse positive negative negative to (+) negative
(erythropoiesis)
M7 negative negative
ALL <1%+ coarse positive < 5 % negative negative polar positive (T)
AUL negative negative negative negative negative
(positive = focal)

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Leukemia diagnostics
Classification of immature-cell leukemia

FAB classification of acute leukemia

In approximately 5 % of leukocytes neoplasias with infiltration of bone marrow and release into the blood stream, it is
not possible to differentiate between immature lymphatic leukemia with negative PAS reaction and immature myeloid
leukemia of the peroxidase -1- type (>/= 5 % POX positive blasts). These cases must be placed into a special group,
cytochemical undifferentiated leukemia. Sometimes it is possible with the aid of an additional characteristic acid
phosphatase reaction and particularly with the aid of immunological determination methods, to reach a fine diagnosis,
usually indicating acute lymphatic leukemia.

In this connection attention is drawn to the increasing use of a leukemia classification developed by a French-
American-British group of hematologists under the leadership of Bennet (1976), the so-called FAB classification of
acute leukemia. It is mainly based on the assessment of comprehensive cytomorphological criteria, the cytochemical
findings being allocated only a secondary function.

In accordance with this FAB classification, acute leukemia is divided into 2 main groups with 3 or 6 subgroups.
The one main group includes acute lymphoblastic leukemia (ALL) with the subtypes L1 to L3, and the other main group
contains acute myeloid leukemia (AML) or acute non-lymphoblastic leukemia (ANLL) with the high inhomogeneous
subgroup M1 to M6 including erythroleukemia and megakaryocytic leukemia see table FAB classification
on the next page. More recently, the megakaryocytic FAB M7 has been differentiated from FAB M6 erythroleukemia.

Nevertheless, even with the FAB classification there is still a not inconsiderable percentage (approx. 3 %) of little
differentiated leukemias which cannot easily be placed in the L2 or M1 subgroup. These morphological
and cytochemical non-classifiable cases of acute leukemia are combined according to a new definition in the
subgroup M0.

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 Leukemia diagnostics
Classification of immature-cell leukemia

FAB classification of acute leukemia is in correlation to the cytomorphological and cytochemical differentiation
characteristics on which they are based.

FAB-Classification Characteristic Cytochemical main reaction Incidence (%) Overall abbreviation

Acute lymphoblastic leukemia (ALL)

L1 small cells (child. L.) PAS, AP 65 ALL
L2 mixed cells, often undiff. PAS + undiff. 30 ALL, AUL
L3 coarse cells, Burkitt type PAS 5 ALL

Acute myeloid leukemia (AML) or acute non-lymphoblastic leukemia (ANLL)

M1 myeloblastic, immature POX-1 14 AML (immature)
M2 myeloblastic, mature POX-2, POX-3, Sudan Black 30 AML (maturing)
M3 promyelocytic, hypergranular POX-3, Sudan Black 6 AProl
M4 myelomonocytic POX-EST, NASDCI 33 AMMoI
M5 monocytic EST 14 AMoL
(A = immature, B = mature)
M6 erythroblastic, megakaryocytic PAS (erythrobl.) 3 AEL

The two most commonly used classification schemata for AML are the older French-American-British (FAB) system
and the newer World Health Organization (WHO) system.

45
Leukemia diagnostics
Myelodysplastic syndrome

Myelodysplastic syndrome
Myelodysplastic syndromes (MDS) are diseases that increase with the increase of age population. Thus demographic
development indicate significant rise in the number of these disease. Enzyme cytochemical staining techniques can be
used in hematological tests for the differentiation of MDS. Beside iron staining, naphthol AS-D chloroacetate esterase
(NASDCL) are important methods for MDS diagnosis, as enzyme pattern abnormalities suggestive of MDS can be
visualized by both positive and negative reaction.

In refractory anemia, more then 15 % of all nucleated red blood cells are in the bone marrow ringed sideroblasts.
Ringed sideroblasts are nucleated red cell precursors which on light microscopy have at least five granules of
hemosiderin. The granules are stained blue with the Berlin blue stain/Prussian blue stain for iron. In refractory anemia
with excess of blasts (RAEB) may be seen ringed sideroblasts.

MDS diagnosis types: 1. Refractory anemia (RA)

2. Refractory anemia with ringed sideroblasts (RARS, D6)
3. Refractory anemia with excess blasts (RAEB)
4. Chronic myelomonocytic leukemia (CMML)
5. Refractory anemia with excess blasts in transformation (RAEBT)

Refractory anaemia (Ringed sideroblasts)

Tabulae haematologicae,
Dr. med. Georg F. Riedler and Dr. med. Raoul Zingg

Iron granules in
small heaps

Ringed sideroblast
Sideroblast

Coarse iron
granules

Ringed sideroblast

Reticular cell
with iron
Sideroblast
Double nucleated
ringed sideroblast

03
Leukemia diagnostics

46
 Leukemia diagnostics
HEMATOGNOST Fe

HEMATOGNOST Fe
Staining kit for the detection of free ionic iron Sample preparation
(Fe3+) in cells
In the Prussian blue reaction, ionic iron (Fe3+) not Smear specimens
bound to the heme structure reacts with potassium Please use thin, air-dried blood or bone
hexacyanoferrate(II) in hydrochloric solution. It marrow smears that have been stored not
precipitates as an insoluble complex salt in the blood, longer than three days
bone marrow, or tissue cells and thus localizes free The smears must be dried in air for at least
cellular iron. 30 minutes and at most 4 hours and be
fixed with methanol
4 Fe3+ + 3 K4Fe(CN)6 = Fe4[Fe(CN)6]3 + 12 K+ Fix the air-dried blood and bone marrow 3 min
smears in methanol
As a measure to achieve the best possible visual Air-dry
differentiation of the iron deposits in the cytoplasm,
this is counterstained with nuclear fast red solution, Histological specimens
resulting in a tender pink color. Deparaffinize the sections in the typical manner and
rehydrate in descending alcohol series

Material
Only fresh, native blood or bone marrow smears should Reagent preparation
be used as the starting material for all stains. The use of Staining solution* for smears and tissue sections
When using the 60-mL Hellendahl cell (with extension),
e.g. EDTA as anticoagulant significantly reduces the
equal volumes are mixed:
peroxidase reaction. In any case, it is not recommended
Reagent 1
to add any anticoagulant substances.
Potassium hexacyanoferrate(II) solution: 30 40 mL
Paraffin sections should be approximately 5 6 m from
Reagent 2
the tissue specimen.
Hydrochloric acid: 30 40 mL

Use only freshly prepared solutions. The staining solution

must be discarded after each staining procedure.

The staining solution* used for counterstaining

(Reagent 3) is ready-to-use, dilution of the solution is
not necessary.

47
Leukemia diagnostics
HEMATOGNOST Fe

Procedure Staining histological specimens in the 60-mL

Carrying out the Prussian blue reaction at 37C yields a Hellendahl cell
sharper definition of the stain precipitates. Slide with histological specimen
Steps Time
Staining smear specimens in the 60-mL Hellendahl cell 1. Staining solution* 20 min
The slides should be allowed to drip off well after (mixed 1+1 from reagents 1 and 2)
the individual staining steps, as a measure to avoid any 2. Distilled water rinse out carefully
unnecessary cross-contamination of solutions. 3. Reagent 3 (nuclear fast red solution)* 5 min
4. Distilled water rinse
Slide with fixed, air-dried smear 5. Ethanol 70 % 1 min
Steps Time 6. Ethanol 70 % 1 min
1. Staining solution* 20 min 7. Ethanol 96 % 1 min
(mixed 1+1 from reagents 1 and 2) 8. Ethanol 96 % 1 min
2. Distilled water rinse 9. Ethanol 100 % 1 min
3. Reagent 3 (nuclear fast red solution)* 5 min 10. Ethanol 100 % 1 min
4. Distilled water rinse 11. Xylene or NEO-CLEAR 5 min
5. Air-dry (e.g. over night or at 50C in the 12. Xylene or NEO-CLEAR 5 min
drying cabinet) 13. Mount the NEO-CLEAR-wet slides with
If necessary mount with aqueous mounting agent Neo-Mount or the xylene-wet slides
(e.g. Aquatex) and cover glass with Entellan new and cover glass

Result
Cell type Color
Free iron (Fe3+) intensive blue granules
Cell nuclei pale red
Cytoplasm tender pink

Bone marrow smear, HEMATOGNOST Fe

03
Leukemia diagnostics

48
 Leukemia diagnostics
HEMATOGNOST Fe

Evaluation Histology
With the binocular microscope (100x, oil immersion) Increased values of serum ferreting indicate
1,000 2,000 cells of a blood smear are differentiated hemochromatosis. Such a suspect has to be confirmed
in a darkened room. The Berliner blue reaction is by liver biopsy and determination of the liver iron
characterized by an intensive turquoise blue color. content. Regularly, a semi-quantitative determination
The siderocytes are determined in of the counted with the Berliner blue reaction is sufficient.
erythrocytes. When differentiating bone marrow Predominantly affected are parenchyma and epithelial
smears, 100 nucleated red cell precursors should be cells of the cystic duct. In tissue sections, concentrations
counted. Besides the quantitative determination of the of ionic iron as low as 0.002 g can be detected with the
sideroblasts, the type and abundance of the iron deposits Prussian blue reaction.
must also be considered (see below).

Normal range
I. In the peripheral blood the normal range
for siderocytes in adults is given as 0 3 .
For newly born babies it is 3 17 .
II. Normal range for sideroblasts in bone marrow
is 20 40 %.

Information to pathological changes

in cellular iron content

Increase
Sideroachrestic and hemolytic anemia, pernicious Ordering information
anemia, lead poisoning, spleen ectomy. Product Package size Cat. No.
Severe overload of an organism with iron causes HEMATOGNOST Fe 1 pack 1.12084.0001
detection of so called ring sideroblasts. In this case, the (for 8 staining batches)
iron deposits are coarse-grained and arranged wreath- Kit content:
shaped in the cytoplasm of red cell precursors. Reticular - Reagent 1:
cells are also Fe3+ positive. Potassium hexacyanoferrate(II) solution (250 mL)
- Reagent 2: Hydrochloric acid (250 mL)
Decrease - Reagent 3: Nuclear fast red solution (2 x 500 mL)
Lack of iron causes a decrease of sideroblasts. Reticular
cells are nearly free from detectable iron deposits.

49
Immersion and mounting
Oil immersion | Mounting with Aquatex

Oil immersion Mounting with Aquatex

Immersion media for microscopy have nearly identical indices as glass. Specimen which were processed according to an enzymcytochemical
Immersion oils practically eliminate light beam deflection so that the method should be preserved with an aqueous mounting agent.
effectiveness of the lens is considerably increased. The refractive index Aquatex is recommended. As soon as the preparation has dried, apply
is around 1.5 and the differences for the convenience of the application an adequate quantity of Aquatex (2 3 drops), to permit homogeneous
is based on the different viscosities. distribution over the entire smear. Apply a clean cover glass avoiding
the inclusion of air bubbles. After embedding leave the preparation to
Application lie horizontally for about 20 30 min until it has set. The slide can then
When microscoping, first locate the part of the dry specimen to be already be immersed in immersion oil and assessed or placed vertically,
investigated. Swing the lens holder away, place a drop of immersion oil in a collection.
on the specimen at the point to be observed and return the lens to
their original position. When finished clean the lens and the specimen Stained slides prepared in this way maintain their colors for months.
with ethanol. A slight yellowing of the mounting agent or small hair-line cracks in the
preparation do not affect the use or the stain.

Ordering information Ordering information

Product Package size Cat. No. Product Package size Cat. No.
Immersion oil for microscopy 100 mL 1.04699.0100 Aquatex 50 mL 1.08562.0050
500 mL 1.04699.0500
Immersion oil acc. to ISO 8036, 100 mL 1.15577.0100
mod. for microscopy
Oil of cedar wood for microscopy 100 mL 1.06169.0100

50
 Storage and documentation
Entellan and Neo-Mount

Entellan and Neo-Mount

A non-aqueous/permanent mounting medium is recommended. As
soon as the slide is completely dried, a few drops of a non-aqueous/
permanent mounting medium are brought up on the preparation. Avoid
air bubbles under the cover slip when the slide is covered. After drying
time of 20 30 min, the smear can be examined under the microscope
and stored in an archive. The so preserved preparation remains color
stable for a minimum of 10 years.

Ordering information
Product Package size Cat. No.
Entellan 500 mL 1.07960.0500
Entellan new 100 mL 1.07961.0100
500 mL 1.07961.0500
1L 1.07961.1000
Entellan new for cover slipper 500 mL 1.00869.0500
DPX new 500 mL 1.00579.0500
Canada balsam 25 mL 1.01691.0025
100 mL 1.01691.0100
Neo-Mount 100 mL 1.09016.0100
500 mL 1.09016.0500

04 05
Immersion/Mounting Storage

51
Blood cell counting

52
 Blood cell counting
Introduction

Blood particles will be counted with automated methods mostly nowadays. There could
be reasons to count by the manual methods in counting chambers especially when
the automats are out of order, when the number of thrombocytes are very low or for
special application as counting in liquor or body effusion or when cell culture are
prepared. In our microscopy portfolio are available 2 products for manual used methods.

Page
06 | Content
Blood cell counting with
- Brilliant Cresyl Blue solution and
Brilliant Cresyl Blue ZnCl2 Certistain 56
- Trks solution 57

06
Blood cell counting

53
Blood cell counting
Brilliant Cresyl Blue solution | Brilliant Cresyl Blue ZnCl2 Certistain

Brilliant Cresyl Blue solution

Staining of reticulocytes in blood with Brilliant Cresyl Procedure
Blue Zinc Chloride Double Salt Certistain Method Counting under the microscope
for the visualization of erythrocyte regeneration by Count the reticulocytes per 1,000 erythrocytes with
counting of reticulocytes oil immersion under the microscope following a
Use for the measurement of substantia meandering pattern. In order to avoid confusion when
granulofilamentosa (ribonucleoproteins) with fresh, counting it is advisable to place a reticulocyte counting
non-fixed, young erythrocytes (supravital staining). Four grid subdivided into small squares (or a square paper
stages of substantia granulofilamentosa maturation can diaphragm) in one of the two eyepieces.
be distinguished depending on the stage of reticulocyte In peripheral blood the development stages III and IV
development: coiled skein (I), incomplete network are found most commonly. When stained with brilliant
(II), complete network (III) and granular form (IV). In cresyl blue they display a dark blue network and dark
peripheral blood the development stages III and IV are blue dots.
found most commonly. When stained with brilliant cresyl
blue they display a bluish black network or bluish black Result
dots. The reticulocyte count is expressed in relation to 1,000
counted erythrocytes (i.e. as 0/00). If the erythrocyte count
Sample material is low, then the absolute reticulocyte count /l is used.
Venous blood, in exceptional cases capillary blood
Calculation
Preparation Reticulocyte count = E/l x R (0/00)
[cells/l]
Brilliant Cresyl Blue solution Dissolve 1.5 g Brilliant 1,000
Cresyl Blue ZnCl2 Certistain in 100 mL physiologic E = erythrocyte count | R = reticulocyte count
saline solution (0.85 % NaCl), and filter. Keep as a
saturated stock solution. Normal range
Brilliant Cresyl Blue working solution Dilute the Patient 0
/00 reticulocyte count/l
Brilliant Cresyl Blue stock solution 1:80 to 1:200 with Adults 5 15 25,000 75,000
physiologic saline solution. For the best dilution make Newborn babies 20 60 100,000 300,000
a test before.
Single tests Draw 20 l blood and 20 l Brilliant Cresyl
Blue solution into a hemoglobin pipette and fill into
a small sealable container. Mix thoroughly and after
about 30 min prepare a thin smear.
Tests in series Prepare thin smears of Brilliant Cresyl
Blue solution on microscope slides using a glass rod. Ordering information
Air-dried slides prepared in this way can be stored for Product Package size Cat. No.
2 3 weeks. For reticulocyte counts smear a small drop Brilliant Cresyl Blue 100 mL 1.01384.0100
of blood quickly over the stain layer, and immediately solution
place the still wet preparation in a moist chamber Brilliant Cresyl Blue 25 g 1.01368.0025
(Petri dish with damp filter paper). Leave for 5 10 min ZnCl2 Certistain
and then allow to dry in air.

54
 Blood cell counting
Trks solution

Trks solution
Reagent for manual leukocyte counting Result
Leukocyte counting is a routine method. The basis of The results are expressed as the means of the duplicate
all counting methods is the dilution and preparation of determinations.
a blood sample of known volume. The erythrocytes are
hemolyzed by the acetic acid of Trks solution and the Calculation
leukocytes are stained by the dye contained. The required Leukocyte count = x 10 10 (dilution 1:10)
cell type in a defined volume is counted and the number 4
of cells per microliter of blood is then calculated. Leukocyte count = x 25 [cells/l]
x = total number of cells counted in the 4 corner squares
Sample material
Anticoagulant venous blood, in exceptional cases Normal range
capillary blood Patient Leukocyte count/l
Adults 4,000 9,000
Preparation School children 5,000 12,000
Filling the pipette Small children 6,000 15,000
Draw blood into the leukocyte pipette up to the 1.0 Infants 7,000 17,000
mark, and then draw Trks solution up to the 11 mark. Newborn babies 10,000 30,000
The dilution is 1:10.
A dilution of 1:20 can also be used (draw blood up to
the 0.5 mark and Trks solution up to the 11 mark).
Mix blood and Trks solution carefully, leave for
maximally 1 h.
Filling the counting chamber
Discard the first 3 drops and fill the counting chamber.

Procedure
Counting under the microscope
Counting is performed with a x 10 objective,
in older microscope models the condenser should be
lowered and its front lens swung out.
Count the leukocytes in the 4 large corner squares,
the sides of which each measure 1 mm.
It is advisable to determine the counts in duplicate;
the results should not differ by more than 15 %. Ordering information
Product Package size Cat. No.
Trks solution 100 mL, 500 mL 109277

Required auxiliaries
Neubauer counting chamber
Leukocyte pipette (with white mixing bead)

06
Blood cell counting

55
References

Literature

Abbrederis, K. Klinische Relevanz zytochemischer Befunde bei differenzierten

myelogenen Leukmien des Erwachsenen.
Fortschritte der Medizin 101, 1732 (1983)
Auer, J. Some hitherto undescribed structures found in the large lymphocytes
of a case of acute leukaemia. Am. J. Med. Sci. 131: 1002, 1906
Baake, M., Gilles, A. Hmatologie, Theorie und Praxis fr med. Assistenzberufe.
GIT-Verlag, Darmstadt 1994
Bain, B. J. Leukaemia Diagnosis a Guide to the FAB classification,
Grower Medical Publishing, London, 1990
Begemann, H., Rastetter, J. Atlas der klinischen Hmatologie, 3. Auflage, Springer-Verlag, Berlin
Heidelberg New York 1978
Begemann, H., Rastetter, J. Klinische Hmatologie, 4. Auflage 1993,
Thieme Verlag, Stuttgart, New York
Bennet, J. M., Catovsky, D., Proposals for the classification of the acute leukaemias.
Daniel, M. T. et al. French-American-British (FAB) Co-operative Group. Br. J. Haematol. 33:
451, 1976
Bennet, J. M., Catovsky, D., Daniel, Proposals for the classification of the myelodysplastic syndromes.
M. T., Flandrin, G., Galton, D. A., Br. J. Haematol 51: 189-199, 1982
Gralnick, H. R., Sultan C.

56
 References
Literature

Bennet, J. M. Classification of the myelodysplastic syndromes.

Clin Haematol 15: 909-922, 1986
Boll, I., Heller, S. Praktische Blutzelldiagnostik
Springer-Verlag, Berlin Heidelberg New York, 1991
Catovsky, D., Greaves, M. F., Pain, C. Acid-phosphatase reaction in acute lymphoblastic leukaemia.
et al. Lancet 1: 749, 1978
DeJong, M. C. Esterase staining of human peripheral blood mononuclear cells.
J. Immunol. Methods 26: 193, 1979
Ehrlich, P. ber die spezifischen Granulationen des Blutes.
Arch. Anat. Physiol., Physiologische Abteilung (Suppl.) 571, 1879
Grusovin, G. D., Castoldi, G. L. Characterization of blast cells in acute nonlymphoid leukaemias by
consecutive cytochemical reactions. Acta Haematol. 55: 338, 1976
Huber, H., Lffler, H., Faber, V. Methoden der diagnostischen Hmatologie.
Springer-Verlag, Berlin Heidelberg New York, 1994
Kaplow, L. S. Histochemical procedure for localizing and evaluating leucocytes alcaline
phosphatase activity in smears and marrow. Blood 10: 1023, 1955
Kass, L. Leukemia. Cytology and Cytochemistry.
Lippincott Company, Philadelphia Toronto, 1982
Lffler, H. Einige Kriterien und praktische Hinweise zur Klassifizierung unreifzelliger
Leukmien. Inn. Med. 2, 219 (1975)
Miltrou, P. S., Lnger F. Atlas der Knochenmark-Neoplasien, Tumorzentrum Rhein-Main e.V.,
Klinikum der J. W. Goethe Universitt Frankfurt/Main, 2001
Queisser, W. (Hrsg.) Das Knochenmark.
Thieme-Verlag, Stuttgart 1978
Riedler, G. F., Zingg, R. Tabulae haematologicae.
Editiones Roche, F. Hoffmann-La Roche & Co. A. G., Basel, Schweiz, 1976
Schmalzl, F., Abbrederis, K., Zytochemische Klassifizierung der akuten Hmoblastosen.
Braunsteiner, H. Lab. med. 1, 99 (1977)
Schumacher, H. R., Garvin, D. F., Introduction to laboratory hematology and hematopathology.
Triplett, D. A. A. R. Liss, New York, 1984
Steward, C. A. Leukocyte alkaline phosphatase in myeloid maturation.
Pathology 6: 287, 1974
Undritz, E. Hmatologische Tafeln Sandoz, z. Auflage.
Eigenverlag der Sandoz AG, Basel 1972
www.aum.iawf.unibe.ch/VLZindex.htm Slide gallery of 2000 blood and bone marrow smears

07
References

57
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