- Serum alkaline phosphatase (ALP) exists in different isoenzyme forms that originate from various tissues like liver, bone and leukocytes. Measuring specific isoenzymes can help identify abnormal increases of total ALP and determine their tissue of origin. - Neutrophil alkaline phosphatase (NAP) is an isoenzyme found in leukocytes. Abnormal NAP levels can indicate various inflammatory conditions and cancers. Semi-quantitative assays are used to measure NAP in serum. - Differentiating between liver-derived ALP, bone ALP and other isoenzymes is important for correctly diagnosing diseases affecting those tissues that cause high total ALP levels, such as liver disease

1. SERUM ALKALINE PHOSPHATASE ACTIVITY AND
ITS ISO-ENZYMES IN INFLAMMATORY
SYNDROMES
Aurelian Udristioiu, Emergency County
Hospital Targu Jiu, Clinical Laboratory, Romania.
Fax: 40253210432; Phone; 40723621414; E-
mail: < aurelianu2007@yahoo.com >

2. Different biochemical forms of alkaline phosphatase (ALP) are
called iso-enzymes.
Neutrophils may be an important source of increased serum
alkaline phosphatase, named neutrophil alkaline phosphatase
(NAP). NAP in Leukemoid Reactions must be distinguished from
other types of serum alkaline phosphatase enzyme, such as bone
alkaline phosphatase (BAP) and bile alkaline phosphatase (bile-AP),
increased in pathological conditions.
The present study describes the indirect evidence that high levels
of ALP in serum may explain the high level of serum alkaline
phosphatase by increasing BAP and NAP in patients with benign
and malignant diseases of bone and inflammatory syndromes.
In the liver, hepatic alkaline phosphatase (HAP) is found on the
edges of cells that join to form bile ducts that drain bile from the liver
into the intestine, where it is necessary to help digest fats in the diet.
HAP is increased in acute and chronic liver diseases.
When a person has high levels of serum ALP and the doctor is
not sure why, for to discovery of causes, he can prescribes
determination Semi-Quantitative Cyto-Chemical NAP.

4. • Total Serum ALP
• Normal values of serum for total ALP there are different in
function of ages: 120-700 U/L for children 1-15 years, 90-190U/L for
adult males and 85-165 U/L for adult females.
• Higher values, past limits of laboratory references, meet in
pathology of liver as acute and chronic hepatitis, hepatic cirrhosis,
biliary obstruction and stasis after 24 hours, malign processes,
colangists, benign and malign diseases of bones,
hyperparathyroidism, duodenal ulcer and in metastases.
• The children have higher ALP because of their bones which are
growing. ALP is often very high during the “growth spurt" which
occurs at different ages in males and females, eating some drugs,
specially used in treat psychiatric problems.

5. Serum iso-enzymes ALP from Liver
• When a person has evidence of liver disease, very high ALP
level can tell to doctor that the person `s bile ducts are somehow
blocked. Often ALP is high to persons which have a cancer that has
spread to liver or bone and doctors can further testing to see if this
has happened.
• If other liver tests, such total bilirubin (TBIL), aspartate amino-
transferase (AST), or alanine - amino-trasferase (ALT) are also
high, usually the ALP is coming from the liver.
• In this case and enzyme gamma-glutamil-transferase (GGT), that
is secreted in the same place with HAP, is high. HAP from liver
represents 60% amount from total serum ALP.

6. • -Bile ALP
• When the bile ducts are blocked, usually by gallstone or by
cancer, ALP and bilirubin may be increased more than AST or ALT.
In the hepatitis, ALP usually much less elevated than AST and ALT.
Also Bile-ALP is increased in cole-stasis, colangio-carcinoma,
primary tumor of liver and metastases of liver.
• -Placentary ALP
• The ALP levels can be increased in Pregnancy of three trimesters
and also in placentary malignancy, (ALP Reagan), by high level
placentary ALP type.
• - Like placentary ALP, which can measured after warm of serum to
56 C*grade time of 10 minutes, is increased in more forms of
cancers.
• The intestinal Izo-enzyme ALP
• Intestinal ALP is increased in cirrhosis and blood group O or B
segregated .The “intestinal” form of ALP is determined by its
phenylalanine sensitivity.
• -Duodenal ALP has normal value until 90 U/L. This type of ALP is
higher in cirrhosis of liver, intra-liver colestasis, enteritis and chronic
hemo-dialysis.

7. • Bone Alkaline Phosphatase in Serum (BAP).
• BAP is a glycoprotein that is found on the surface of osteoblast
cells. BAP has been shown to be a sensitive and reliable indicator of
bone metabolism. If calcium and phosphate measurements are
abnormal, usually the ALP is confirmed as originally from the bones
in some bone diseases such as a disorder called Paget`s disease or
in cancer with bone metastasis.
Method of Measurements of BAP
• By an immune-enzymatic assay is utilized a mouse monoclonal
antibody specific to BAP, paramagnetic particles coated with
antibodies and complex Ag -Ac is measured with illuminometers.
Normal values of BAP there are until 90 U/L, (30% of total ALP)
•

8. • Current Methods of Measurement of ALP from serum:
• 1. Cinetic-Colorimetric Method has as principle the activity of
enzyme ALP in basic medium which p-nitrophenyl phosphate
molecules. Finally results p-nitrophenol and an inorganic phosphate
molecule. It use only fresh serum or plasma, delivered on heparin
without hemolysis.
• Ions of magnesium accelerate activity of enzyme. Increased
absorbance per units of time (yellow intense color) is proportionally
with activity of alkaline phosphatase to 405 nanometers wavelength.
For the type of Photometers with filters must accomplished the
calibrations using a Standard.

9. • 2. The dry Chemistry uses the “Vitros ALKP Slide which is a
multilayered analytical element coated on a polyester support.
• The p-nitrophenol which absorbs light of wavelengths in the region
of 400 nm, diffuses into be underling layer of Vitros slide and it is
monitored by reflectance of spectrophotometers. The rate of change
in reflection density is converted to enzyme activity.
•
• Reference interval of manufactures comprises the central 95% of
results from an interval study of 273 apparently healthy, adults from
a working population, (154 females and 119 males) and has the
normal range 36-126 U/L.ALP.
• Some metabolic components and Drugs that have significant light
absorbance in the region 400 nanometers can cause a special
interference (bilirubin, methotrexate, nitrofurantoin. A compilation of
this information is available in the literature.

10. • 3. . Serum NAP ( neutrophil alkaline phosphatase) activity in serum can
be measured by the method of Japanese Society of Clinical Chemistry, in
which ALP its-enzymes are separated electrophoretically with Titan 3
supporting media (Helena Laboratories).
• Neutrophil Alkaline Phophatase (NAP), an enzyme closely associated
with neutrophil granules, is ready measured by a quantitative chemical
method or semi-quantitative histo-chemical method.
• NAP appears to be restrictive in bands of segmented neutrophil
granulocytes. Its delivery from lysozome in cytoplasm is controlled by 2
genes of 21 chromosomes.
• On colored smears, in microscopic field, sites with enzymatic activity
NAP will appear as dark-blue grants or black grants in cytoplasm of
granulocytes.
• The interpret of reaction will be made in function of score to 100
segmented granulocytes after amounts and intensity of colored grants in
scale 1-4.
• The score of Leukocyte Alkaline Phosphatase will be the product
between number of counted cells and values of percent (%).
Normal score are 60-100 values.

11. • Technical Method of Laboratory:
• Kit SIGMA:
• -Sample-Fixation with solution alcohol-phenol 9/1
concentration,
• -Incubation with glicero-phosphate substrate,
• -Ad nitrate of cobalt 2% concentration,
• -Wash slide,
• -Ad solution sulfur ammonium acid Calcium black
sulfur,
• -Coloration with May Grunwald Giemsa.

13. • DISCUSSIONS;
• In microscopy field, on smear, granulocytes display predominant
toxic granulations, Dohle bodies, and in serum there is elevated
NAP.
• -In mature granulocytes from CML, NAP decreases until “0”, and
than appears after treatment with chemotherapy.
• -Permits the differentiation, in case of leukocytosis, between CML,
where NAP is absent and PV where NAP is increased past normal
interval.
• -Permits the differentiation of secondary poliglobuly, with NAP
decreased or lower score with PV when NAP is very increased.
• -Can to differentiate an Acute Leukemia un-treated with cortyzol
drugs:
• A) In Mieloblastic Acute Leukemia, in mature neutrophiles NAP zero
or lowly.
• B) In Limphoblastic Acute Leukemia, in mature neutrophiles NAP is
decreasd.
• C) In Hanry Cells Leukemia, with severe neutropenia, NAP is very
high.

14. • In Non-Hodgkin Acute Lymphoma NAP is decreased but in
Hodgkin Lymphoma has very high score.
• To children, the score LAP is increased in CML. In the
Threesomya 21 –Mongolism, NAP is increased because of surplus
of chromosomes.
• The enzyme NAP must be examined only in segmented and un-
segmented neutrophils and eosinophils. The basophil is negatives
NAP and not must be counted in the level of score ALP.
• The enzyme NAP permits the physical-pathological
understanding of neutrophils which though in microscopy field
appear normally but these have a modified chemical content and
emphasis the functionality of leukocytes.

17. • CONCLUSION
•
• -Direct evidence of iso-enzymes ALP is can be done by
the biochemical and immunochemical properties of ALP,
especially in the serum to patients with high numbers of
leukocytes and we have proposed to assess laboratory
results for to help clinicians in correct decisions to
hospitalized patients, especially in the Point-of-Care
Department.
• - We consider that this our objective must be in the
attentions of all Laboratories.