cy to chemistry

8/22/2019 Cy to Chemistry

1/25

.


2/25

Study of chemical elements found in cells This elements can either be:

1. Enzymatic e.g. peroxidases

2. Non-enzymatic

e.g. lipids and glycogen


3/25

Acceptable specimens Smears and imprints made from:

1. Bone marrow

2. Lymph nodes

3. Spleen4. Peripheral blood

Enzymatic smear specimen: fresh, newly obtainedspecimen are preferred

Non Enzymatic specimen: Periodic Acid Schiff(PAS) and Sudan Black remains stable even after amonth


4/25

Acceptable fixatives should contain:1. Alcohol (methanol, ethanol)

2. Acetone

3. Formaldehyde


5/25

MYELOPEROXIDASE (MPX) Enzyme found in the primary granules of PMNs,

Eosinophils and to certain extent Monocytes

(-) Lymphocytes

Differentiates Blasts from Acute MyeloidLeukemia (AML) from Acute LymphoblasticLeukemia (ALL)

Principle:

Myeloperosidase oxidizes the substrate in thepresence of hydrogen peroxide black to redbrown


6/25

MYELOPEROXIDASE (MPX)Interpretation:

AML (w/o maturation, with maturation,promyelocytic leukemia) is 80% positive with MPX

Auer rods strongly positive to MPX Auer rods are found in leukemic blasts and

promyelocyte

Monocytes are MPX negative to weakly positive

Lymphoblasts are negative; ALL 3% areperoxidase positive


7/25

MYELOPEROXIDASE (MPX) Important: Blast cells be only used as a

differentiation among the acute leukemias


8/25

SUDAN BLACK B

Differentiation of acute myeloid leukemiafrom acute lymphoblastic leukemia

More sensitive for early myeloid cells

Principle: Sudan Black stains lipids such as sterols, neutral

fats and phosphilipids

SB is soluble to lipids

Lipids are found on:

1. the primary and secondary granules of PMNS

2. lysosomal granules of monocytes


9/25

SUDAN BLACK B

Interpretation Granulocytes are positive from the

myeloblast throughout the maturationseries

The staining capacity is directlyproportional to cell growth, maturationand the asquisition of primary andsecondary granules


10/25

SUDAN BLACK B


11/25

ESTERASES Used to differentiate the myeloblasts from the

neutrophilic series from the cells of themonocytic origin

Nine isoenzymes of esterases are present inleukocytes

Substrates esters commonly used:1. Non-specific: a-naphthyl butyrate, a-naphthyl

butyrate2. Specific: Naphthol AS-D chloracetate esters

Specificity: staining of specifically myelocyticcells only


12/25

ESTERASES

Principle:

Esterases hydrolyzes an ester.

At the site of any enzyme activity, if it

reacts with a naphthol compund, andcombines with a diazonium salt, it willform a brightly colored compund

Diazonium salts: pararosaniline,

hexazotized new fuschin or fast blue


13/25

ESTERASES Interpretation


14/25

PERIODIC ACID-SCHIFF Diagnosing Acute Lymphocytic Leukemia and other

erythroid tyoe of Acute myeloid leukemia

Principle:

Periodic acid oxidizes glycogen, mucoproteins band

other high molecular weight carbohydrate intoALDEHYDE

Aldehyde + colorless schiff reagent ---bright redpink

The intensity of stain is directly proportional to thenumber of aldehyde compounds produced


15/25

PERIODIC ACID-SCHIFF PAS Stain can either be:

1. Fine and diffuse

2. Coarse and granular


16/25

PERIODIC ACID-SCHIFF Interpretation

Granulocytes are PAS positive

Megakaryocytes has a finely diffuse staining

Platelets are intensely red pink

Erythrocyte precursor does not stain

ALL: Lymphoblast may stain coarse or fine or mixed

ERYTHROID: (+) coarse and granular


17/25

Condition

MPX SBB NASDA ANBE ANAE PAS F VIIALL - - - -/+ -/+ Varied -

AML + + + - - Varied -

AMML + + + +

diffuse

+

diffuse

Varied -

AMoL - +- - +diffuse

+diffuse

Varied -

Erythro-

leukemia

* * * - - +;blotchy

inpronor-moblast

-

MegakaryocytciLeukem

ia

- - - - +localize

d

-/+localize

d

+

* = (+) in myeloblast (-) in normoblast


18/25

FACTOR VIII ANTIBODIES

Megakaryoblastic leukemia

(+) result is from the reaction withmonoclonal or polyclonal antibidies against

Factor VIII-related antigen


19/25

LEUKOCYTE ALKALINE PHOSPHATASE (LAP)

Differentiates Chronic Myelogenus leukemiaand leukemoid reaction

Leukemoid reaction is seen in severe

infections Principle:

LAP is seen in the membrane of secondarygranules of neutrophil

Substrate naphthol AS-BI phosphate is hydrolyzed+ dye (fast red violet, fast blue BB) ---produces acolored precipitate at the site of LAP enzyme


20/25


SCORE No. Of Cells Score x No. Of cells0 20 0

1 45 45

2 25 50

3 5 15

4 5 20

Total 100 130=LAP Score

Please refer to page 403 for the example of LAP scoring


21/25


Reminders in scoring LAP Subjective method

Two slides be read by 2 different clinical laboratorytechnologist

The scores done by the the 2 technologist shouldagree by 10%

If nota 3rd should be obtained

Eosinophils should be identified from the

neutrophils Laboratory values should be established by each

laboratory


22/25


Interpretation of LAP Score Normal 20-100

Untreated Chronic Myelogeous Leukemia: decrease

Leukemoid reaction: high normal to increase

Low LAP Scores:

Paroxysmal Nocturnal Hemoglobinuria

Sideroblatic Anemia

Myelodysplastic Disorders

High LAP Scores: Pregnancy in the third trimester

Polycythemia Vera


23/25

FINDING SCORENormal 20-100

Chronic Myelogenous Leukemia 100

Polycythemia Vera 100-200Secondary Polycythemia 20-100


24/25

ACID PHOSPHATASE (Tartrate Resistant)

Detection of Hairy Cell Leukemia

All cells contain 7 non-erythroid isoenzymes:

Isoensymes: 0, 1, 2, 3, 3b, 4 and 5

Hairy cells is isoenzyme 5 positive Principle:

Acid Phosphatase + AS-BI phosphoric acid + dye(fast garnet GBC) ---Red ppt

Red ppt + L-(+) Tartaric acid ---all isoenzymesare inhibited EXCEPT FOR ISOENZYME 5

TRAP PHENOMENA: ISOENZYME 5 IS RESISTANT TOTARTRATE


25/25

Cell Type Without tartrate With TartrateLymphocyte + -

Hairy Cells + +

cy to chemistry

Documents