-
HiMedia Laboratories Pvt. Ltd.www.himedialabs.com
- CORPORATE OFFICE -A-516, Swastik Disha Business Park, Via
Vadhani Indl Est, LBS Marg,
Mumbai - 400 086, India.Tel : +91-22-6147 1919 / 2500 3747 | Fax
: +91-22-6147 1920 / 2500 5764
Email : [email protected], [email protected]
- OVERSEAS OFFICES -
USA & CanadaHiMedia Laboratories LLC, 107 W Dorothys Way,
Lincoln University, West Chester,
Pennsylvania 19352, USA.Tel : +1-484-734-4401 | Fax :
+1-484-734-4402
Email : [email protected]
EuropeHiMedia Laboratories GmbH, Marie-Curie-Str. 3, 64683,
Einhausen, Germany.Tel : +49 6251 989 24 26 | Fax : +49 6251 989
24 27
Email : [email protected]
Lite
ratu
re C
ode
: TL2
35_2
/ Pe
riphe
ral B
lood
Kar
yoty
ping
Sol
utio
ns B
roch
ure
/ 071
8
Peripheral Blood Karyotyping Solutions
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Cytogenetic analysis involves six principal steps, culturing
cells, metaphase arrest, harvest of metaphase chromosomes,
chromosome preparation, banding and staining using a special
protocol, and analysis by light microscopy or karyotype assisted
computer analysis. The discovery that colchicine (or Colcemid)
pretreatment resulted in mitotic arrest and that treatment of
arrested cells with a hypotonic solution improved the yield and
quality of metaphases spreads.
Cytogenetic study is considered a mandatory investigation in
newly diagnosed leukemia owing to its usefulness in disease
diagnosis, classification and prognostication. The vast majority of
recurrent chromosomal rearrangements associated with leukemia were
originally identified by cytogenetic analysis, which remains the
gold standard laboratory test since it provides a comprehensive
analysis for abnormality on the entire genome.
Although other cells of the body can be cultured for cytogenetic
studies, peripheral blood cells are used most often due to several
advantages such as easy collection procedure and lesser testing
time (3 days).
Our objective is to make quality diagnostic tools for making
your cytogenetic analysis convenient, easy and accurate.
HiKaryoXL™ Karyotyping Media HiKaryoXL™ media are a series of
ready-to-use media developed for short term in vitro culture of
peripheral blood lymphocytes for cytogenetic studies. These media
are based on either RPMI 1640 or Nutrient Mixture F-10 Ham and are
further supplemented with L-Glutamine, FBS, Penicillin,
Streptomycin and Sodium bicarbonate. They are supplied with or
without PHA-M to suit the convenience of users.
1
It’s All About Understanding GeneticsQuality controlEvery lot of
Medium & reagent is performance tested to ensure consistence,
accuracy, reproducibility and superior performance.
Performance TestMitotic index : Calculated as percentage of
cells arrested at metaphase
Sterility TestMycoplasma : Detection by PCR
Bacterial, Yeast and Fungi : As per current edition of USP
Physicochemical testspH and Osmolality
Lot-to-lot consistencey assured
Manufactured under GMP, ISO 13485 and ISO 9001 certified
facility
CE Marked for IVD (98/79/EC)
Avoid repeated Freeze-thaw Use 10ml convenient pack
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2
Mitotic StimulatorsLymphocytes from peripheral blood are
mitotically inactive and have to be stimulated with a mitogen. In
presence of a mitogen, small lymphocytes undergo a process known as
transformation, in which the cell enlarges and the staining
properties of the nucleus change. Such a transformed cell is
capable of cell division.
Lymphocytes in purified preparation or in whole blood can be
stimulated with different mitogens such as Phytohemagglutinin
(PHA), Concanavalin A, Pokeweed mitogen (PWM) etc.
Phytohemagglutinin (PHA) and Concanavalin A affect primarily the T
cell population while Pokeweed mitogen (PWM) affects the B cell
population.
HiKaryoXL™ PHA-M SolutionThe most common mitogen used for the
stimulation of cell division in lymphocyte cultures is
Phytohemagglutinin (PHA). PHA causes small T lymphocytes to
transform to lymphoblasts and enter mitosis. PHA acts by causing a
marked increase in RNA synthesis within the first 24 hours.
Lymphocytes produce interleukin-2 (IL-2) or lymphocyte growth
factor which further stimulates mitosis. DNA synthesis is low
during the first 30 hours of culture but it increases steadily
between 30 and 60 hours.
The use of PHA-M as a mitogen helps to obtain lymphocyte that
are actively dividing, thus yielding analyzable mitotic chromosome
spreads. PHA-M is a lectin extracted from red kidney bean Phaseolus
vulgaris. The protein consists of two subunits, a leucoagglutininin
(PHA-L) and an erythroagglutinin (PHA-E). PHA-M is the mucoprotein
form and is most commonly used in cytogenetics laboratories.
Product Name Code Packing
HiKaryoXL™ PHA-M Solution w/ 0.1 mg per ml PHA-M in sterile
tissue culture grade water
TCL061-10ML 10ml 5x10ml
HiKaryoXL™ PHA-M Solution w/ 1 mg per ml PHA-M in sterile tissue
culture grade water
TCL071-10ML 10ml 5x10ml
PHA-M (Phytohemagglutinin-M) Cell Culture Tested
TC209-10MG TC209-25MG TC209-4X25MG
10mg 25mg 4x25mg
HiKaryoXL™ Karyotyping Reagents PHA-PPhytohemagglutinin (PHA-P)
is a lectin isolated from red kidney beans Phaseolus vulgaris. It
is purified by affinity chromatography. PHA-P has a molecular
weight of 115kDa. The lectin PHA-P consists of five glycoproteins
that are tetrameric structures made up of two subunits PHA-E
(erythroagglutinin) and PHA-L (leucoagglutinin). PHA-E has a low
mitogenic activity and a high erythroagglutination activity whereas
PHA-L has a high mitogenic and leucoagglutinating activity, but
very low erythroagglutinating activity. PHA-E is not blood group
specific but agglutination can be inhibited by certain
oligosaccharides.
Product Name Code Packing
PHA-P (Phytohemagglutinin-P) Cell Culture Tested
TC226-5MG TC226-5X5MG
5mg 5x5mg
Concanavalin AConcanavalin A (Con A) is a glycoprotein isolated
from Jack bean (Canavalia ensiformis). Con A specifically binds
with specific terminal sugar residues like a-mannose and
a-galactose structures found in sugars, glycoproteins and
glycolipids. It agglutinates red blood cells and complexes with
blood group substances, immunoglobulin, glycopeptides and
carcinoembryonic antigens. Hence it is widely used in hormone
receptor studies, mitogenic assays and for characterizing normal
and malignant cells. It is also used to initiate mitogenesis in T
lymphocytes by stimulating the energy metabolism of thymocytes.
In neutral and alkaline solutions, concanavalin A exists as a
tetramer consisting of 4 subunits of 26.5kDa each. In acidic
solutions (pH below 5.0), concanavalin A exists as a dimer.
Product Name Code Packing
Concanavalin A Cell Culture Tested
TC220-25MG TC220-100MG
25mg 100mg
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HiKaryoXL™ Colcemid® SolutionColcemid® also known as
demecolcine, is related to colchicine but it is less toxic. It
depolymerises microtubules and limits microtubule formation
(inactivates spindle fibre formation), thus arresting cells in
metaphase and allowing cell harvest and karyotyping to be
performed.
Product Name Code Packing
HiKaryoXL™ Colcemid® Solution w/ 10µg per ml Colcemid in Hanks'
Balanced salt solution
TCL074-20ML TCL074-100ML
20ml 100ml
HiKaryoXL™ Colcemid® Solution w/ 10µg per ml Colcemid in
Phosphate Buffered Saline
TCL133-20ML TCL133-100ML
20ml 100ml
Colcemid® Demecolcine Cell Culture Tested
TC566-5MG TC566-10MG TC566-50MG
5mg 10mg 50mg
Colcemid® is a registered trademark of Ciba - Giegy Corp.
Stains
Giemsa StainG-banding is a technique used in cytogenetics to
produce a visible karyotype by staining condensed chromosomes.
Banding can be used to identify chromosomal abnormalities because
there is a unique pattern of light and dark bands for each
chromosome. The metaphase chromosomes are treated with trypsin and
stained with Giemsa.
Product Name Code Packing
Giemsa Stain Solution TCL083-100ML TCL083-500ML
100ml 500ml
Giemsa stain Cell Culture Tested
TC232-5G TC232-25G
5gm 25gm
Mitotic InhibitorsProper techniques for harvesting dividing
cells and preparing slides for chromosomal analysis are critical
for attaining the quality needed for correct karyotype analysis.
Chromosomes become structurally and numerically distinct only
during the metaphase stage of cell division. Mitotic inhibitors
like Colchicine and Colcemid are used to collect the cells at this
stage for cytogenetic analysis. Mitotic inhibitors disrupt mitotic
spindle fibers and free the chromosomes from metaphase plate,
accounting them to spread out inside the cell. The lack of spindle
fibers also blocks the anaphase so the mitotic index is effectively
increased. Mitotic inhibitors also cause chromosome contraction.
The degree to which chromosomes contract depends on the
concentration and also the time that the cells are exposed to the
mitotic inhibitors.
HiKaryoXL™ Colchicine SolutionColchicine, an alkaloid isolated
from the plant Colchicum autumnale, is a microtubule-depolymerizing
agent that has been used to arrest cells at metaphase. Arresting of
cells in metaphase allows an increased yield of mitotic cells for
analysis. Colchicine inhibits microtubule polymerization by binding
to tubulin, one of the main constituents of microtubules.
Availability of tubulin is essential to mitosis, and therefore
colchicine effectively functions as a “mitotic poison” or spindle
poison.
Product Name Code Packing
HiKaryoXL™ Colchicine Solution w/ 10µg per ml Colchicine in
Phosphate Buffered Saline
TCL062-20ML 20ml
Colchicine Cell Culture Tested
TC030-500MG TC030-1G TC030-10G
500mg 1gm 10gm
3
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4
Related Reagents
Potassium Chloride Solution, 0.075MA hypotonic solution of
potassium chloride is used in blood lymphocyte chromosome
preparation. The hypotonic treatment causes the cells to swell and
aids in the release of the intact chromosomes. The KCl hypotonic
treatment is important for swelling of the cells and adequate
spreading of chromosomes on the slide.
Product Name Code Packing
Potassium chloride Solution, 0.075M
TCL040-100ML 100ml
Trypsin-EDTA SolutionsG-banding requires pretreatment of
chromosomes with trypsin to partially digest the chromosomes which
are then stained with Giemsa stain. Each homologous chromosome pair
has a unique pattern of G-bands, enabling recognition of particular
chromosomes.
Product Name Code Packing
Trypsin-EDTA Solution 10X w/ 2.5% Trypsin (1:250), 0.2% EDTA in
0.85% normal saline w/o Phenol red
TCL070-100ML TCL070-500ML
100ml 5x100ml 2x500ml
Trypsin Solution for G-Banding w/ 0.025% Trypsin in Dulbecco's
Phosphate Buffered Saline
TCL122-100ML 100ml 5x100ml
Gurr Buffer SolutionGurr buffer is a used for G-banding of
chromosomes by Giemsa staining for cytogenetic analysis.
Product Name Code Packing
Gurr Buffer Solution pH 6.8 TL1139-500ML 500ml
Hoechst 33258 & Hoechst 33342Hoechst, a bis-benzimidazole
derivative compound, is a DNA intercalator. It preferentially binds
to adenine-thymine (A-T) regions of DNA. It is excited by
ultraviolet light at around 350 nm and emit blue/cyan fluorescence
around an emission maximum at 461 nm. Hoechst stain may be used on
live or fixed cells. It is also used to stain the metaphase
chromosomes with much brighter fluorescence and different banding
pattern. Hoechst stain shows minimal background fluorescence and
slow quenching. Ability of Hoechst 33342 to permeate the cells is
about 10 times higher than Hoechst 33258.
Product Name Code Packing
Bisbenzimide (Hoechst 33258) Cell Culture Tested
TC225-25MG TC225-100MG
25mg 100mg
PHA-P (Phytohemagglutinin-P) Cell Culture Tested
TC226-5MG TC226-5X5MG
5mg 5x5mg
DAPI- 4’, 6-Diamidino-2-phenylindoleDAPI
(diamidino-2-phenylindole) is a fluorescent stain that binds
strongly to the DNA. It is rapidly taken up by the cellular DNA
because of its high cell permeability. It selectively binds to the
minor groove of double stranded DNA. The excitation maximum for
DAPI bound to dsDNA is 358 nm, and the emission maximum is 461 nm.
DAPI can be used for both fixed and live cell staining, though the
concentration of DAPI needed for live cell staining is generally
much higher than for fixed cells.
Product Name Code Packing
DAPI dihydrochloride (4’,6-Diamidino-2-phenylindole
dihydrochloride) Cell Culture Tested
TC229-5MG TC229-10MG
5mg 10mg
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5
Troubleshooting
Problem Possible cause Solution
Chromosome quality
Contracted chromosomes Prolonged treatment with mitotic
inhibitorTreat the culture with mitotic inhibitor for recommended
time
Chromosomes not well spread across the slide/ overlapping
chromosomes
Non-uniform drying of the slide Avoid blowing, dropping from
height & flaming
Cell density too highDilute the suspension with fixative and
repeat dropping
Scattered chromosomes Cells burst open during slide makingEnsure
that the addition of hypotonic solution and fixative is performed
gently
Slide quality
Particulate matter and debris appearing on slide
Cell clumps and debris present
Mix the cell pellet uniformly and gently to break the clumps and
repeat dropping
If clumps do not break, allow the clumps and debris to settle,
remove cell suspension to a separate tube and repeat dropping
Procedural problems
Dark brown irreversible clumping of cells
Harsh addition of hypotonic solution and / or fixativeEnsure
gentle addition of hypotonic solution and fixative
Cell growth
No cell growthInappropriate growth conditions
Check temperature and percentage of CO2 in the incubator
Blood sample is not fresh Always use fresh blood
G-Banding
No G-bands seen on chromosomes
Insufficient trypsin treatmentOptimize the duration of trypsin
exposure to get optimum banding
Prolonged stainingDO NOT expose the slide to Giemsa stain for
longer duration. Optimize the timeof exposure to Giemsa stain
Fuzzy G-bands banding Over trypsinization Optimize the duration
of trypsin exposure
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6
Blood Collection
Culture
Harvest
Analysis
Peripheral blood lymphocytes were cultured in 3 different lots
of HiKaryoXL™ Medium (product code: AL249A). After 72 hours,
karyotyping procedure was performed. Slides were stained using
Giemsa stain and number of mitotic cells /slide were counted.
Chromosome spread from normal periferal blood cells cultured in
HiMedia's HiKaryoXL™ Media for 72 hours at 100X magnification.
Product Name Code Packing
Media containing PHA-M as mitogen
HiKaryoXL™ RPMI Medium w/ L-Glutamine, FBS, PHA-M, Penicillin,
Streptomycin and Sodium bicarbonate
AL165A-10ML AL165A-100ML
50x10ml 5x100ml
HiKaryoXL™ Nutrient Mixture F10 Medium w/ L-Glutamine, FBS,
PHA-M, Penicillin, Streptomycin and Sodium bicarbonate
AL169A-10ML AL169A-100ML
50x10ml 5x100ml
Media containing PHA-P as mitogen
HiKaryoXL™ RPMI Medium w/ L-Glutamine, FBS, PHA-P, Penicillin,
Streptomycin and Sodium bicarbonate
AL249A-10ML AL249A-100ML
50x10ml 5x100ml
Media without mitogen
HiKaryoXL™ RPMI Medium w/ L-Glutamine, FBS, Penicillin,
Streptomycin and Sodium bicarbonate w/o PHA-M
AL173A-10ML AL173A-100ML
50x10ml 5x100ml
HiKaryoXL™ Nutrient Mixture F-10 Ham Medium w/ L-Glutamine, FBS,
Penicillin, Streptomycin and Sodium bicarbonate w/o PHA-M
AL185A-100ML 5x100ml
457
LOT 1
Num
ber o
f mito
tic c
ells
/slid
e
Lot to lot consistency of HikaryoXL™ RPMI Medium (AL249A)
LOT 2 LOT 3
369429
-
HiMedia Laboratories Pvt. Ltd.www.himedialabs.com
- CORPORATE OFFICE -A-516, Swastik Disha Business Park, Via
Vadhani Indl Est, LBS Marg,
Mumbai - 400 086, India.Tel : +91-22-6147 1919 / 2500 3747 | Fax
: +91-22-6147 1920 / 2500 5764
Email : [email protected], [email protected]
- OVERSEAS OFFICES -
USA & CanadaHiMedia Laboratories LLC, 107 W Dorothys Way,
Lincoln University, West Chester,
Pennsylvania 19352, USA.Tel : +1-484-734-4401 | Fax :
+1-484-734-4402
Email : [email protected]
EuropeHiMedia Laboratories GmbH, Marie-Curie-Str. 3, 64683,
Einhausen, Germany.Tel : +49 6251 989 24 26 | Fax : +49 6251 989
24 27
Email : [email protected]
Lite
ratu
re C
ode
: TL2
35_2
/ Pe
riphe
ral B
lood
Kar
yoty
ping
Sol
utio
ns B
roch
ure
/ 071
8
