Hema Procedures

NAME: Lorelie Montales Dejaño PROFESSOR: Dr. Maria Cecilia B. Asilo CLASS NO: 13 DATE OF SUBMISSION: January 20, 2011

DITHIONITE TEST A. PRINCIPLE.

Erythrocytes are introduced into a phosphate buffer solution containing a reducing agent and hytic agent. The red cells are lysed and the hemoglobin is reduced. Reduced sickling types of hemoglobin are insoluble in phosphate buffer and turbidity results. On addition of urea, hemoglobin S dissolves. B. REAGENTS

• Stock phosphate buffer solution. Dissolve 160.48 grams anhydrous potassium dihydrogen phosphate (KH2PO4), AR, and 28.88 grams anhydrous potassium monohydrogen phosphate (K2HPO4) and AR in a 1-liter volumetric flask containing 500 ml of distilled water. Dilute to 1-liter with distilled water.

• Dithionite reagent. Add 20.0 grams dithionite (Ha2S404 2H20) and 0.25 grams saponin to a 100-ml volumetric flask. Add 80 ml of stock phosphate buffer solution. Mix well. Dilute to the mark with distilled water. This reagent remains stable under refrigeration at 4°C for approximately 1 month. See figure 6-1.

• Urea (USP). C. PROCEDURE.

1. Pipet 2 ml of dithionite reagent in 12 x 75 mm test tube. 2. Add 0.02 ml of well-mixed anticoagulated blood

(collected in EDTA). 3. Mix the contents and al1a.v to stand at room temperature

for 5 minutes (see figure 6-1). 4. After 5 minutes examine the tube for turbidity against a

lined reader. Hemoglobin 5, if present, produces turbidity in the tube .

Figure 6-1 D. SOURCES OF ERROR.

1. The use of 10 x 75 mm test tubes could cause a false negative result. 2. The dithionite reagent has a limited stability. The freshness of this reagent must be

checked with positive and negative controls. The test should show the blue-pink color of reduced hemoglobin and adequate lysis of erythrocytes.

3. Unstoppered tubes containing dithionite reagent decompose when left out at room temperature.

NAME: Lorelie Montales Dejaño PROFESSOR: Dr. Maria Cecilia B. Asilo CLASS NO: 13 DATE OF SUBMISSION: March 3, 2011

ERYTHROCYTE SEDIMENTATION RATE

PRINCIPLE: The distance of which red blood cells have fallen if left at room temperature for an hour. MATERIALS

o Venipuncture kit o Wintrobeʼs tube and Westergrenʼs tube o Rack o Timer o Pipette

PROCEDURE: 1. Collect 5 ml of blood using sodium citrated tube 2. Pipette 1 ml of blood and transfer it to either Westergrenʼs or Wintrobeʼs tube 3. Place the tube in a rack and make sure it is vertically placed because slight tilting

can increase erythrocyte sedimentation rate. 4. After an hour, examine the tubes and get the erythrocyte sedimentation rate. FACTORS THAT INCREASE ESR:

a. Slanting the tube b. Presence of acute phase

reactants c. Increase levels of

immunoglobulin d. Menstruation and pregnancy e. Plasma proteins f. Wrong use of anticoagulant g. Fibrinogenemia

FACTORS THAT DECREASE ESR:

1. Excess anticoagulant 2. Presence of poikilocytes

NORMAL VALUES:

Adult male: 0-15 mm Adult female: 0-20 mm


ACIDIFIED-SERUM TEST (HAM TEST)

PRINCIPLE: The patient’s red cells are exposed at 37 °C to the action of normal or the patients own serum suitably acidified to the optimum pH for lysis (pH 6.5-7.0). SAMPLE:

The patient’s red cells can be obtained from defibrinated, heparinized, oxalated, citrated or EDTA blood, and the test can be satisfactorily carried out even on cells which have been stored at 4°C for up to 2-3 weeks in ACD or Alsever’s solution, if kept sterile. The patient’s serum is best obtained by defibrination, for if in PNH it is obtained from blood allowed to clot in the ordinary way at 37°C or at room temperature it will almost certainly be found to be markedly lysed. Normal serum should similarly be obtained by defibrination, but serum derived from blood allowed to clot spontaneously at room temperature or at 37°C can be used. Normal serum known to be strongly lytic to PNH red cells is to be preferred to patient’s serum, the lytic potentiality of which is unknown. However, if the test is positive using normal serum it is important, particularly if the patient appears not to be suffering from overt intravascular hemolysis, to obtain a positive result using the patient’s serum, in order to exclude HEMPAS. The activity of a single individual’s serum also varies from time to time and it is always important to include in any test, as a positive control, a sample of known PNH cells or artificially created “PNH-like” cells.

The sera should be fresh, i.e. used within a few hours of collection. Their lytic potency is retained for several months at -70°C, but at 4°C, and even at -20°C, they deteriorates within a few days. TECHNIQUE: 1. Deliver 0.5 ml samples of fresh normal serum, group AB- or ABO- compatible

with the patient’s blood, into six (three pairs) of glass tubes. 2. Place two tubes at 56°C for 10-30 minutes in order to inactivate the

complement. 3. Keep the other two pairs of tubes at room temperature. 4. Add to the serum in two of the tubes one-tenth volumes (0.05 ml) of 0.2 mol/l

HCL. 5. Add similar volumes of acid subsequently to the inactivated serum samples. 6. Place all tubes in a 37°C water-bath.

7. Wash samples of the patient’s red cells and of control normal red cells (compatible with the normal serum) twice in 9.0 g/l NaCL.

8. Prepare 50% suspensions in the saline. 9. Add one-tenth volumes of each of these cell suspensions (0.05 ml) to single

tubes containing unacidified fresh serum, acidified fresh serum and acidified inactivated serum, respectively.

10.Mix the contents carefully and leave the tubes at 37°C for one hour. 11.Centrifuge. In case of quantitative measurement of lysis * Add 0.05 ml of each cell suspension to 0.55 ml of water so as to prepare a standard. * Retain 0.5 ml of serum to use as a blank. * Deliver 0.3 ml volumes of the supernatants of the test and control series of cell-serum suspensions, and of the blank serum and of the lysed cell suspension equivalent to 0% and 100% lysis, respectively, into 5 ml of 0.4 ml/L ammonia or Drabkin’s reagent. * Measure the lysis in a photoelectric colorimeter using a yellow-green (e.g. Ilford 625) filter or in a spectrophotometer at a wavelength of 540 nm. INTERPRETATIONS: If the test cells are from a patient with PNH: * They will undergo definite, although, as already mentioned incomplete lysis in the acidified serum. * Very much less lysis, or even no lysis at all, will be visible in the unacidified serum. * No lysis will be brought about by the acidified inactivated serum. * The normal control sample of cell should not undergo lysis in any of the three tubes.

In PNH 10-50% lysis is usually obtained, when lysis is measured as liberated hemoglobin. Exceptionally, there may be as much as 80% lysis or as little as 10%. The red cells of a patient who has been transfused will undergo less lysis than before transfusion, because the normal transfused cell, despite circulation in the patient, behave normally. In PNH, it is characteristic that a young cell (reticulocyte-rich) population, such as the upper red cell layer obtained by centrifugation, undergoes more lysis than the red cells derived from mixed whole blood.


HEAT INSTABILITY TEST PRINCIPLE:

When hemoglobin in solution is heated the hydrophobic van der Waals bonds are weakened and the stability of the molecule is decreased. Under controlled conditions unstable hemoglobins precipitate while stable hemoglobin remains in solution. SAMPLE:

Samples taken into any anticoagulant are satisfactory. EDTA is the most convenient. Cells freed from clotted blood can also be used if necessary. REAGENT:

Tris-HCL buffer, pH 7.4, 50 mmol/l. Tris 6.05 g, water to 1 liter. Adjust the pH to 7.4 with concentrated HCL. METHOD: 1. Preparation of lysate: (Lyse 1 volume of washed packed cells in 4 volumes of

lysing reagent prepared as follows: • g EDTA, tetrasodium salt. • 0.7 g potassium cyanide (KCN). • Water to 1 liter.

2. Add 0.2 ml of lysate, freshly prepared to 1.8 ml of buffer. Include a positive (Hb F) and a negative (Hb A) control of the same age as the test sample.

3. Such a lysate will not keep for more than 1-2 days at 4 °C as it tends to gel. If necessary it can be frozen at –20 °C for up to 1 month. Avoid repeated freezing and thawing).

4. Place the tubes in a water-bath at 50 °C for 120 min. examine the tubes at 60, 90, and 120 min for turbidity and fine flocculation.

INTERPRETATION & COMMENTS:

The normal control may give minimal cloudiness at 60 min but a major unstable hemoglobin will have undergone marked precipitation at 60 min and gross flocculation at 120 min.


KLEIHAUER-BETKE/ ACID ELUTION FOR FETAL HEMOGLOBIN/K-B

The Kleihauer-Betke ("KB") test, Kleihauer-Betke ("KB") stain or Kleihauer test, is a blood test used to measure the amount of fetal hemoglobin transferred from a fetus to a mother's bloodstream[1]. It is usually performed on Rhesus-negative mothers to determine the required dose of Rho(D) immune globulin (RhIg) to inhibit formation of Rh antibodies in the mother and prevent Rh disease in future Rh-positive children.

The Kleihauer-Betke (KB) test, also known as acid elution for foetal haemoglobin , is where blood is drawn from the mother's vein and tested for the presence of foetal cells, in order to determine the amount of Rh immune globulin an Rh-negative woman must receive to prevent her from developing antibodies, or abnormal proteins, against her foetus. PROCEDURE

Blood is drawn from a vein and analysed for the presence of foetal cells with a special staining technique known as the Kleihauer-Betke method . The greater the foetal blood leakage into the mother's circulation, the greater the amount of Rh immune globulin the mother must receive. INDICATION

Staining of postpartum maternal blood for identification of percentage of fetal cells present. Determine possible fetal maternal hemorrhage in the newborn, aid in diagnosis of certain types of anemia in adults; assess the magnitude of fetal maternal hemorrhage; calculate dosage of Rh immune globulin to be given. PHYSIOLOGY The KB test is helpful in distinguishing some forms of Thalassemia from hereditary persistence of fetal hemoglobin. The hereditary persistence of fetal hemoglobin reveals a uniform distribution of fetal hemoglobin in each red cell. By calculating the amount of fetal blood contamination, the evolution of anti-D antibodies in the post partum woman and subsequent hemolytic disease of the new born can be prevented. NORMAL RANGE : full-term newborns: Hb F cells are > 90%; normal adults Hb F cells are < 0.01%. TEST METHOD: Acid elution test.


LUPUS ERYTHEMATOSUS PREPARATION (L.E. PREP.)

In patients suffering from systematic lupus erythematosus, a specific characteristic cell, termed the L. E. cell, is found in the bone marrow and peripheral blood, when the smears are prepared according to a specific procedure. This L.E. cell phenomenon is caused by an immunoglobulin present in the patientʼs plasma, called the L. E. factor. REAGENTS AND EQUIPMENT

1. Wire sieve and pestle 2. Petri dish 3. Wintrobe sedimentation rate tubes, three or four. 4. Clean glass slides. 5. Wright stain and buffer. 6. Disposable droppers with long enough to reach the bottom of a Wintrobe tube.

SPECIMEN: Clotted, whole blood, 10 mL PRINCIPLE

Clotted blood is allowed to sit at room temperature for two hours. Forcing it through a sieve then macerates the clot. The trauma, produced when the blood is force through the strainer, causes extrusion of nuclei from the poly-morphonuclear cells. The L.E. factor present in the blood lyses the nuclear material, which is then phagocytized by other neutrophils. This forms the L.E. cell. PROCEDURE

1. Place 10 mL of whole blood in a plain test tube, and allow the blood to clot. 2. ncubate the tube of clotted blood at room temperature, for two hours. 3. Using an applicator stick to hold the clot in the test tube, pour off the serum and discard it. 4. Place the sieve over a perti dish. 5. Place the clot in the sieve. 6. Mash the clot throygh the sieve, using the pestle. 7. Transfer the blood from the Petri dish to three or four Wintrobe sedimentation rate tubes,

using disposable droppers of the proper length. 8. Centrifuge the filled Wintrobe tubes at 2500 RPM for 20 minutes. 9. Remove the serum from each of the tubes, using a disposable dropper. Be careful not to

remove any of the buffy coat (layer of white cells). 10. Using a clean disposable dropper, remove the buffy coat and transfer a small drop to

each of three or four slides, and make smears. 11. Wright-stain the smears and examine for the presence of L.E. cells. Report out as

positive or negative. The characteristic L. E. cell appears as a neutrophil containing a


large spherical body in its cytoplasm. Ordinarily, the nucleus of the neutrophil is pushed to one side of the cell and may appear wrap itself around the ingested material. The L. E. body shows no nuclear structure and stains as a pale purple homogeneous mass. It has a velvetlike appearance. In rare instances, the ingesting cell may be a monocyte or eosinophil. The L. E. body surrounded by neutrophils. These are readily seen using low power (10X) magnification. The tart cell, which may be confused with the L. E. cell, is usually a monocyte which has ingested another cell or the nucleus of another cell. In this case, the ingested material will usually resemble a lymphocyte nucleus or phagocytized material with a definite nuclear pattern. Another form of ingested material found in the tart cell is an intensely stained body termed a pyknotic nucleus. The significance of these cells is not known. The presence in an L. E. preparation does not signify a positive test for systematic lupus erythematosus.

DISCUSSION 1. An alternative method to the above procedure employs the use of 5 mL of whole blood, mixed

with 0.5 mL of dilute heparin. Five glass beads (4 to 5 mm in diameter) are added to the tube. After incubating the blood at room temperature for 30 miinutes, the blood is mixed on a rotator for 30 minutes, and reincubated at room temperature for one hour. The blood is then placed in Wintrobe sedimentation tubes, centrifuged, and blood smears made from the buffy coat, as previously described. This method is considered to be more sensitive than the procedure employing clotted blood. However, the concentration of heparin is important, since high concentrations of anticoagulants are thought to inihibit L. E. cell formation.

2. When examining the blood smear, each of three slides should be studied for approximately ten minutes each before a report is made. The smear should be examined, using the highdry objective (45X) or on low power (10X) when enough experience has been gained. All suspiciuous cells should be examined under oil immersion (100X)

3. The presence of one L.E. cell is not a substantial basis for reporting out a positive result. Several very typical L.E. cells should be seen before a positive report is made.

4. If a patient has severe leucopenia, a false negative result may be obtained due to the decrease in neutrophils present. Therefore, since the L.E. factor is present in the serum, add 5 mL of patientʼs serum to 5mL of washed red cells (type O blood) obtained from a normal individual. Carrry out the test as previously described.

5. Occassionally, false positive results will be obtained in patients having disorders such as rheumatoid arthritisʼ drug sensitivity, and hepatitis.

6. Patients with systemic lupus erythmatosus, who are on adrenocorticosteroid theraphy, may have false negative L. E. tests. Also, L. E. cells are not demonstrated in some people having the active clinical disease.

7. The L.E. factor is also termed an antinuclear factor, since it reacts against cell nucei.


OSMOTIC FRAGILITY PRINCIPLE: The resistance of red blood cells to lysis when subjected to hypotonic solution PURPOSE: 1. To aid diagnosis of hereditary spherocytosis 2. To supplement a stained cell examination to detect morphologic RBC abnormalities MATERIALS:

Test tube rack Test tubes Timer 0.02% NaOH dropper distilled water

PROCEDURE:

1. Get 16 tubes and label the 1st tube on the left with “25”, the 2nd tube with “24” and so on and so forth until the 16th tube

2. Place a drop of blood to each test tube 3. Add 0.02% NaOH to all the test tubes. The number of drops corresponds to the label of the test tubes. 4. Add distilled water. The number of drops corresponds to the difference of the label number from 25 5. Mix the mixture well and stand all the tubes for 1 hour at room temperature 6. Observe for hemolysis after an hour and record the tubes that shows the partial and complete hemolysis

PARTIAL HEMOLYSIS – first tube from left to give pinkish solution and is turbid; tells that some of the red blood cells have lysed. (0.36 – 0.42% NaOH) COMPLETE HEMOLYSIS – First tube to give clear solution and there is precipitate; tells that all red blood cells have hemolyzed. ( 0.26 – 0.32% NaOH)

INTERPRETATION:

The osmotic fragility of fresh red cells reflects their ability to take up a certain amount of water before lysing. This is determined by their volume to surface area ratio. The ability of the normal red cell to withstand hypotonicity results from its biconcave shape which allows the cell to increase its volume by about 70 % before the surface membrane is stretched; once this limit is reached lysis occurs. Spherocytes have an increased volume to surface area; their ability to take in water before stretching the surface membrane is this more limited than normal and they are therefore particularly susceptible to osmotic lysis. The increase in osmotic fragility is a property of the spheroidal shape of the cell and is independent of the cause of the spheroytosis.

Decreased O.F. indicates the presence of unusually

flattened red cells (leptocytes) in which the volume to surface area ratio is decreased. Such a change occurs in iron deficiency anemia and thalassemia in which the red cells with a low MCH & MCV are unusually resistant to osmotic lysis.


SUCROSE LYSIS TEST

PRINCIPLE: The sucrose lysis test is based on the fact that red cells absorb complement components from serum at low ionic concentrations. PNH cells because of their great sensitivity will undergo lysis but normal red cells do not. SAMPLE:

• Patient sample on EDTA. • Clotted sample (ABO compatible with the patient) to have compatible serum as

control. TECHNIQUE: Isotonic solution of sucrose = 0.924 g in 10 ml distilled water (92.4 g/l)(can be stored at 4 °C for up to 2-3 weeks).

* Two test tubes are prepared for the test and the control. * Put the following in these tubes.

Solute / Saline

Compatible Serum Patient’s washed RBCs

(50% suspension) Test 850 ml 50ml 100ml Control 850ml 50ml 100ml 1. Incubate 30 minutes in 37 °C. 2. Centrifuge then examine for hemolysis. 3. If lysis is visible in sucrose containing tube, measure this in a spectrophotometer

(WL 546), using a tube containing serum diluted in saline as a blank and a tube containing 0.1 ml of red cell suspension in 0.9 ml of 0.4 ml/l ammonia in place of the sucrose-serum mixture as a standard for 100% lysis.

INTERPRETATION: • Red cells from some cases of leukemia or myelosclerosis may undergo a small

amount of lysis, almost always <10%; in such cases the acidified-serum test is usually negative and PNH should not be diagnosed.

• In PNH, lysis varies from 10% to 80%, but exceptionally may be as little as 5%. Sucrose lysis and acidified-serum lysis of PNH red cells are fairly closely correlated.

• The sucrose lysis test is typically negative in HEMPAS

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Hema Procedures