HEMA - SPECIAL HEMA TEST MIDTERM

PNH (Paroxysmal Nocturnal Hemoglobinuria) ➢ Screening test

○ Sugar water test ○ Sucrose hemolysis test

PNH (Paroxysmal Nocturnal Hemoglobinuria) ➢ Confirmatory test ➢ Flow Cytometric Analaysis

○ Ham's test

automated and will assess how many CD 55 or 59 is lost in the cell

Flow Cytometric Analaysis

decaying accelerating factor

CD 55

membrane-inhibitor reactive lysis

CD 59

Screening test used for the assessment of PNH.

Sugar Water Test

Sensitive to Paroxysmal Nocturnal Hemoglobinuria but not specific to PNH.

Sugar Water Test

If Sugar Water Test shows a positive result, it is necessary to do a confirmatory test such as

Sucrose hemolysis or Ham test.

Other disease that may be tested in Sugar Water Test

Leukemia or myelosclerosis

Principle: Serum contains complement proteins

Sugar Water Test

Principle: Complement will attach to RBC at sugar solution if no CD 55 or 59 present, which will lead to hemolysis

Sugar Water Test

Solution of Sugar Water Test

Patient's whole blood + sugar water solution

Solution of Sugar Water Test: Room temperature and allow it to stand for

Patient's whole blood + sugar water solution

10 minutes

Positive indicator of PNH

Presence of hemolysis

Negative indicator of PNH

No hemolysis

Confirm the diagnosis of PNH

Ham's Test

Ham's Test also called as

Acidified Serum Test

Ham's Test: RBC without CD 55 and 59 are prone to spontaneous complement mediated hemolysis, especially if the blood pH is slightly (pH?)

acidic

RBC without CD 55 and 59 are prone to ??? especially if the blood pH is slightly acidic.

spontaneous complement mediated hemolysis,

3 types of serum:

normal serum, patient's serum inactivated serum

Inactivated serum heat at ??? to deactivate certain complement proteins such as C4 and C2

56 C

Inactivated serum heat at 56 C to deactivate certain complement proteins such as ?????

C4 and C2

Ham's Test Inactivated serum: add ???? to make RBC susceptible to complement lysis

0.2 N HCl

Positive Result for PNH

Tubes with patient's serum and normal serum will show hemolysis

Tube with inactivated serum will show

no hemolysis (clear supernatant)

Hemolysis occur in normal serum and inactivated serum

CDA type II (HEMPAS)

No hemolysis in patient's own serum but occurs in normal and inactivated serum

CDA type II (HEMPAS)

Mild hemolysis in all of the serum preparations made

Hereditary spherocytosis.

RBC is < 5 um with no central pallor

spherocytosis

Defective α or β chain of spectrin, ankyrin, and protein band 3 and 4.2

Hereditary Spherocytosis

In Hereditary Spherocytosis, defective α or β chain of ??, ??, and protein band ??

spectrin ankyrin protein band 3 protein band 4.2

Macrophage will eat the blebs at the spleen, leading to early destruction

Hereditary Spherocytosis

May use

• EOFT

• Eosin-5'-maleimide binding test

Test for Hereditary Spherocytosis

EOFT: Subject RBC at different concentrations of salt starting from?

1. ???? solution (1.0% NaCl) or ???? solution (0.90-0.85% NaCl)

2. ???? solution (0.0% NaCl)

1. Hypertonic solution (1.0% NaCl) or hypotonic solution (0.90-0.85% NaCl)

to

2. ???? solution (0.0% NaCl)

Normal RBC ○ No lysis:

0.5 % NaCl

Normal RBC ○ Partial hemolysis:

0.40-0.45%NaCl

Normal RBC ○ Complete hemolysis:

0.30-0.35% NaCl

Defective RBC will lyse at ?? solutions

hypotonic

Also an abnormal membrane defect, a genetic abnormality causing deficiency or abnormal structure

Hereditary spherocytosis

Hereditary spherocytosis membrane protein affected:

Spectrin

Disorders with hypochromic RBC

▪ Iron Deficiency Anemia ▪ Thalassemia ▪ Anemia of chronic disorders ▪ Sideroblastic anemia

Disorders with target cells

▪ Thalassemia ▪ Hemoglobinopathies

Tests for RBC metabolism

➢ Fluorescent spot test ➢ Auto hemolysis test ➢ Ascorbate cyanide test

Screening for presence of normal activity of the G-6-PD and Pyruvate kinase in the blood

Fluorescent spot test

1 ml drop of anticoagulated whole blood using either EDTA, Heparin or ACD, is placed to a special filter paper

G6PD -Fluorescence Spot test

Principle ▪ NADP is reduced to NADPH ▪ Presence of the NADPH

Fluorescence Spot test

Positive Result for Fluorescence Spot test (G6PD)

fluoresce

Negative Result for Fluorescence Spot test (G6PD)

No fluorescence

Major enzymes under the EMP pathway. Responsible for production of enough ATP.

Pyruvate kinase

For Pyruvate kinase, blood is placed in a filter paper and mixed with reagent containing ?, ADP, and reduced NADH

Fluorescence Spot test

Blood is placed in a filter paper and mixed with a reagent containing oxidized glutathione (GSSG) and NADPH

Glutathione test

Positive Glutathione Reductase Test

NADPH to NADP

Sufficient Glutathione Reductase (GSSR)

fluoresce for 20 minutes and disappear after

Deficient Glutathione Reductase (GSSR)

fluorescence for more than 1 hour

Heinz bodies stain used

supravital stain

A reducing substance that can oxidize hemoglobin and show the inclusion (heinz bodies)

acethyphenylhydrazine

positive for Acethyphenylhydrazine meaning positive for Heinz bodies

(+) >32% with 5 or more

negative for Acethyphenylhydrazine meaning positive for Heinz bodies

(-) <32% with 5 or more

oxidized form of hemoglobin

Heinz Bodies.

supravital stains used in heinz bodies

methylene blue or brilliant cresyl blue.

Lead to bite cells and cause early destruction (hemolytic anemia)

Heinz bodies

Oxidative Drugs causing Heinz bodies formation

➢ Primaquine - Malaria ➢ Quinolone - Malaria ➢ Phenazopyridine ➢ Nitrofurantoin ➢ Fava beans ➢ Sulfadiazide ➢ Cortrimoxazole ➢ Toluidine blue ➢ Methylene blue ➢ Dapsone

Autohemolysis Test Screen the two most common red cell enzymopathy:

G-6PD deficiency and pyruvate kinase deficiency.

AUTO HEMOLYSIS TEST: Blood specimen is subjected to a warm temperature ranging from

37 C to 40 C for 48 hours.

AUTO HEMOLYSIS TEST: If no hemolysis with glucose reagent

G-6PD deficiency

AUTO HEMOLYSIS TEST: If no hemolysis with ATP reagent

pyruvate kinase deficiency

AUTO HEMOLYSIS TEST: If no hemolysis with glucose reagent = (what auto hemolysis type?)

Auto hemolysis type I

AUTO HEMOLYSIS TEST: If no hemolysis with ATP reagent = (what auto hemolysis type?)

Auto hemolysis type II

AUTO HEMOLYSIS TEST: If no hemolysis with both the glucose and ATP reagent

hereditary spherocytosis.

AUTO HEMOLYSIS TEST: If no hemolysis with both the glucose and ATP reagent = (what auto hemolysis type?)

Auto hemolysis type III

Evaluates the activity of the Hexose monophosphate pathway or Pentose phosphate pathway

Ascorbate Cyanide Test

Ascorbate Cyanide Test: Reagent used

Sodium cyanide and Sodium Ascorbate

One of the known methods used is the Jacob and Jandl

Ascorbate Cyanide Test:

Uses aerated whole blood (oxyhemoglobin, mixed with sodium cyanide and sodium ascorbate.

Ascorbate Cyanide Test

Principle: Sodium ascorbate + oxyhemoglobin produces hydrogen peroxide (inactivated by either catalase or glutathione peroxidase)

shuts down the catalase activity, allowing only the hexose monophosphate pathway to do its job to transfor hydrogen peroxide to a non-toxic substance such as water and oxygen

Sodium cyanide

not hmp but helps in reducing hydrogen peroxide to water and oxygen within the red cells.

Catalase

Sodium cyanide and Sodium Ascorbate forms ?

hydrogen peroxide = blood oxidizes forming brown color

one of the oxidized products produced by the action of hydrogen peroxide which causes the formation of this brown pigment on blood samples.

Methemoglobin

Ascorbate Cyanide Test: Normal pentose phosphate pathway (blood)

blood is red

Ascorbate Cyanide Test: Normal pentose phosphate pathway (hydrogen peroxide)

Hydrogenperoxideisreducedanddeactivated

Ascorbate Cyanide Test: Defective pentose phosphate pathway (blood)

blood is brown

point mutation in amino acid so beta globin is unstable:

Glutamic, 6th amino acid, is replaced with valine

Hgb S

Caused by amino acid substitution resulting to instability of the Hgb molecules which then affects the normal shape of the RBC

Hemoglobinopathies

Screening test for unstable Hgb

○ Heat denaturation test ○ Isopropanol precipitation test ○ Heinz bodies test

Unstable hemoglobin is incubated with 17% isopropanol and incubated (37 C)

Isopropanol Precipitation test

Principle ▪ Nonpolar solvents weakens the bonds of Hgb molecule causing it to precipitate

Isopropanol Precipitation test

Laboratory Test for Unstable Hgb ➢ Isopropanol Precipitation test

Unstable hemoglobin

precipitate at 20 minutes ▪ Faster rate of precipitation formation ▪ Bonds are easily weakened by isopropanol

Hemolysate, mixed with tris buffer solution (7.4 pH), is incubated at 50 C for 2 hours

Heat Denaturation test

Principle ▪ Unstable hemoglobin → heavy precipitate (heat sensitive)

Heat Denaturation test

Hemoglobin concentration of both supernatant will be determined using the formula of ?? method

cyanmethemoglobin method

Beta globin chain is affected at the 6th position of amino acid in the chain

Sickle Cell Anemia (Hgb S)

Sickle cell disease

○ SS - (homozygous) sickle cell anemia ○ AS - (heterozygous) sickle cell trait ○ SC - α2β6Glu-Valβ6Glu-Lys ○ SD-Punjab - α2 β6Glu-Val β121Gln-Glu ○ SO-Arab - α2 β121Glu-Lys ○ S- B thalassemia ○ SE - α2β26Glu-Lys ○ S-heredity persistence fetal Hgb

This polymerization elongates the Hgb and change RBC shape to elongated with pointed structure called

sickle cells or drepanocytes

What triggers sickling of RBC

○ Low oxygen tension ○ Dehydration ○ Low blood pH

Sickle cell formed may be trapped and clogged within the minute vasculature especially of the kidney and spleen and cause occluded within it.

Vaso occlusive crisis

Vaso occlusive crisis could affect various tissues and organs aside from

kidney or spleen like bone, lungs, liver eyes, and CNS.

Screening Test for Sickle Cell

➢ Sodium metabisulfite test ➢ Dithionite solubility test

➢ Also known as the sickling test

Sodium Metabisulfite Test

Sodium Metabisulfite Test: Incubate with ????? reagent

sodium metabisulfite reagent