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Cont’n of Phosphatases Non-Enzymatic Techniques

Leukocyte Alkaline
PAS-Schiff reagent Sudan Black B Toluidine Blue
Phosphatase
Reagents: -colorless soL’n capable of for the demonstration of certain a dye that can bind w/ acid
Fixative: formalin 40mL reacting w/ aldehyde groups in phospholipids and lipoproteins mucopolysaccharides in blood cells to form
Methanol 360mL glycogen, mucoproteins and metachromatic complexes
Store in freezer. other high molecular weight Mechanism of action is uncertain in that it
Tris buffer 0.2M pH9.1 carbohydrates. may be: Ex. granules of both basophils&mast cells are
Trizma base 48.44mg Fixative: formalin-10mL 1. Selective adsorption stromgly metachromatic-stain reddish violet
Distilled H2O 200mL Absolute methanol- 2. A chemical reaction
Adjust pH to 9.1 w/ 1N 90mL 3. Or a combination Usefulness:
HCl Keep Recognition of mast cell diseases and
capped&refrigerated. SBB reaction is similar to the acute or chronic basophilic leukemias.
Substrate soL’n: myeloperoxidase reaction.
Dissolve 0.6g of naphthol-AS- 1% Periodic-kept refrigerated,
BI phosphate in 10mL N,N- should be made fresh every 7 Advantage of SBB over the
dimethylformamide. days myeloperoxidase stain:
1. Its stability to heat and storage
Add 0.2M Tris buffer to 2L. Store Schiff’s rgt: commercially 2. more sensitive to primitive
in refrigerator. prepared. Keep refrigerated. myeloid cells
Colorless, if it turns pink—it must 3. the reagent are not carcinogenic
be discarded
Disadvantages:
Harris hematoxylin 1. Require 1-2 hours staining time
2. its specificity
Ammonia water 3-5 drops conc. 3. increased background staining on
NH4OH in 50mL distilled H2O bone marrow specimen
Incubation Mixture: secondary to the fatty nature of
bone marrow itself Ferric Iron-Prussian Blue Reaction
Working soL’n:
Presence of iron granules in otherwise
50mg of fast blue BBn
dissolve in 50mL of mature, nonnucleated red cells
(siderocytes) may be seen in iron overload
substrate, prepared
fresh&filter before use. or poor iron utilization syndromes

Procedure:
Cold fixatives (4°-10°C)-30
secs Reagents:
Working soL’n-20 minutes Fixative:absolute methyl alcohol
Nuclear fast red-
counterstain-20mins Interpretation:
Bone marrow iron storage is
Controls: usuallyreported as absent, decreased,
Slides taken from pregnant increased or normal
women in their last trimester
Other principal use of the iron stain in
Interpretation: Glycogen is predominant in
leukocytes. hematology is the evaluation of red cell
Count 50 segmented
iron utilization.
neutrophils.
Score staining reaction from 0- + color-bright fuschia-pink
Troubleshooting:
4+
Presence of excessive amounts of
extracellular material that stains + for iron
0=no granules
can be caused by contaminated reagents.
1+=very few granules
2+=moderate # of granules
[-] staining in a + control may be
scattered throughout the cell
secondary to improper pH of the K
3+=numerous granules starting
ferrocyanide.
to coalesce
4+=cytoplasm packed w/
granules

Immuno Cytochemical Techniques


Immunochemistry is defined as the identification of the immunologic phenotype of a given cell population throught the use of specific monoclonal or polyclonal
antibodies against selected cell antigens.
Specimens used: A slide-based technique is advantageous because: =both practical and cost effective
 Cell Suspension 1. it allows for counting of designated cell =no expensive equipment is required
 Paraffin or cryostat section populations = specimen may be as much as 1 week old
 Smears 2. permanent slide can be filed for future
 Imprints review Application of this technique in hema lab has been
primarily for the identification of the cell types
 Cytospin prep
involved in acute or chronic leukemias.
Enzyme Immunocytochemistry (Immunoperoxidase)
 Peroxidase-antiperoxidase (PAP)  Immunoalkaline phosphatase PAP technique uses 3 rgts:
 Plant enzyme used—horseradish peroxidase 1. primary antibody that is specific for the antigen in
Advantages: Advantages: question
1. easily obtainable&stable lack of cross reactivity w/ the 2. secondary AB that will bind to the Fc portion of the 1
2. endogenous peroxidase is easily blocked pseudoperoxidase of AB&carries the PAP complex as a tag
3. variety of chromogens are available to react w/ peroxidase to erythrocytes/granulocytes 3. substrate used to make the peroxidase reaction
form a colored end product visible
inexpensive
Method-depend on the kit commercially available Control-buffy coat preparation made from peripheral blood from healthy individual