SIP

Subject: Blood Banking
Topic: Bone Marrow Transplantation (BMT)

Bone marrow contains immature cells (stem cells), which develops into RBCs, WBCs & platelets

Purpose: To replace unhealthy cells with healthy ones in order to survive

Patients: People with diseases of the blood, bone marrow, or certain types of cancer, (e,g., Hodgkin's disease, mulitple myeloma, leukaemia, etc.)

Conditioning/preparative regimen: Treatment with high doses of chemotherapy or total body irradiation, given immediately prior to a transplant.

Purpose: To help remove the patient's disease prior to the BMT & to suppress immune reactions. It also has an immunosuppressive effect which prevents rejection of the BMT by the recipient's immune system.

If donor's bone marrow doesn not match, it'll treat the patient's body as foreign material to be attacked & destroyed, which results in graft vs host disease (GVHD). This is when patient's own immune system destroy the new bone marrow (graft rejection).

During the period when patient waits for the transplanted bone marrow to migrate to the cavities of large bones, setting up 'engraft' to the stage where it begins to produce normal blood cells, the patient is susceptible to infection & excessive bleeding. Platelets are transfused to patients who are bleeding.

Until the donor marrow engrafts, RBCs & platelets will be needed to keep the hemoglobin level above 9g/dl and platelet count > 20,000/ul. Engraftment takes place in 2 to 4 weeks but may be delayed if complications occur.

As a result of the total body irradiation & chemotherapy required for preparation for transplantation, patients are immunosuppressed & susceptible to GVHD, if transfused with blood components that contains live lymphocytes, which are capable of dividing. Tranfusion related GVHD can be prevented by irradiating all blood components.

Why use irradiated & filtered (leucodepleted) blood products?

To prevent patients from febrile non-hemolytic transfusion reaction due to leucocytes & leucocytes fragments in whole blood or packed cells.

Blood cell gamma irradiator is used for irradiating blood components such as red cell or platelet concentrates. The gamma rays effectively stop cells dividing by mitotic division, preventing lymphocyte proliferation.

Since alloimmunisation is caused by leucocytes contained in the blood components, leucodepleted red cells has been used to minimise the likelihood of inducing alloimmunisation.

The immunosuppression also results in great susceptibility to infectious diseases. Most patients will receive anitbiotics, antifungal agents & intravenous immunoglobulin to help prevent fight infection.

ABO Incompatible Transplants:
ABO incompatible transplants may be done when there is patient antibody directed against donor cells (major incompatibility; e.g., patient O, donor A) or donor antibody directed against the patient's red cells (minor incompatibility; e.g., patient A, donor O).

Example 1:
Patient 1's blood group: A+
Donor 1's blood group: O+

Blood product (irradiated & filtered): Packed cell: O+
Platelet/CSP: Group A+ (1st choice)/Group O+

Problem: Continued production of antibody by the new donor lymphocytes while there are original patient's cells continuing to circulate. Thus, the group O donor cells may begin to produce anti-A while the orginal patient A red cells remains, leading to hemolysis.

Solution: This can be lessened by red cell exchange transfusion if necessary.

Group A Recipient with Group O Donor:

Anti-A	Anti-B	Anti-AB	A Cells	B Cells	O Cells	Interpretation
+	-	+	-	+	-	Group A
+/-	-	-	-	+	-	Incomplete Group A
-	-	-	+/-	+	-	Incomplete Group O
-	-	-	+	+	-	Complete Group O

ABO blood grouping of transfused patient:
For incomplete group A, there will be a mixed field reaction when tested with anti-A. Some of the red cells are agglutinated while others are not. The anti-sera will appear to be cloudy due to some free red cells. This occurs when the patient's blood group (A+) is slowly switching to the new donor's blood type (O+).

Example 2:
Patient 2's blood group: O+
Donor 2's blood group: A+

Blood product (irradiated & filtered): Packed cell: O+
Platelet/CSP: Group A+ (1st choice)/Group AB+

Problem: When ABO-incompatible marrow is to be transfused into a patient with circulating antibodies against the donor's ABO type, hemolysis of the red cells in the marrow can be expected.

Prevention: By processing the marrow to remove the red cells.

Following transplant & after engraftment, the patient will become A+ but cannot begin to receive group A red cells until the circulating anti-A has disappeared. Anti-A persists longer than anti-B. Thus, even after transfusion of type A marrow, the patient continues to receive type O red cells containing additonal anti-A that can slow down the appearance of type A red cells.

If delayed red cell engraftment is occurring, the titer of anti-A should be determined & a DAT done to determine if high levels of circulating anti-A are hemolysing newly forming A red cells.

In making the decision when to convert to the new donor ABO type for red cell transfusion, we use the crossmatch to determine the absence of circulating ABO antibody incompatible with the patient's original type. The patient's serum should be tested in the antiglobulin phase to ensure the absence of free ABO-incompatible antibody.

When the ABO antibody to donor type is undetectable & DAT is negative, this is a satisfactory indicator of the safety to begin transfusion the new donor ABO type red cells.

Group O Recipient with Group A Donor:

Anti-A	Anti-B	Anti-AB	A Cells	B Cells	O Cells	Interpretation
-	-	-	+	+	-	Group O
+/-	-	+/-	-	+	-	Incomplete Group A
+	-	+	-	+	-	Complete Group A

dorothy png
tg01

Staining and staining problems

Staining and staining problems

There is 2 main types of stains that is used most commonly. They are the Romanowsky and Papanicolaou(and it's derivatives). Romanowsky-type stain are more rewarding and practical, and readily available in practice situations. Example of such stains are Wright's stain, Giemsa stain, Diff-Quik. Romanowsky-type stain are inexpensive, readily available to practicing veterinarian, easy to prepare, maintain and use. They stain organism and cytoplasm of cells very well.

Smears are first air dried, preserving(fix) the cells, and cause them to adhere to the slide so they do not fall off during the staining procedure. Diff-Quik does not undergo the metachromatic reaction. As a result, granules of some mast cells do not stain, When mast cell granules do not stain, the mast cells may be misclassified as macrophages. This can lead to confusion in examination of some mast-cell tumors. Variation between different Romanowsky-type stains should not cause a problem once the evalutator has become familiar with the stain used routinely.

The procedure recommended for each stain should be followed generally. However, adaptations is to be applied according to the thickness of smear. Thick smear, higher protein concentration, longer stain time. Vice-versa for thin smear.

Poor stain quality often confuse both the novice and the experienced cytologist. Most staining problem can be avoided if the following precaution are taken:

• Use new slides, fresh and well filtered(if needed) stain, and fresh buffer(if needed)


• Stain cytologic preparations immediately after drying


• Take care not to touch the surface of slide or smear at any time.
  
  

The table below shows some common problems and solution.

Problem	Solution
Excessive Blue Staining
Prolonged stain contact	Decrese staining time
Inadequate wash	Wash longer
Specimen too thick	Make thinner smears
Stain, diluent, buffer or wash water too alkaline	Check with pH paper and correct pH
Exposure to formalin vapors	Store and ship cytologic preps separate from formalin containers
Wet fixation in ethanol or formalin	Air dry smears before fixation
Delayed fixation	Fix smears sooner
Surface of the slide was alkaline	Use new slides
Excessive Pink Staining
Insufficient staining time	Increase staining time
Prolonged washing	Decrease duration of wash
Stain or diluent too acidic	Check with pH paper and correct pH; fresh methanol may be needed
Excessive time in red stain solution	Decrease stain time in red solution
Inadequate time in blue stain solution	Increase time in blue stain solution
Mounting coverslip before preparation is dry	Allow preparation to dry completely before mounting coverslip
Weak Staining
Insufficient contact with one or more of the stain solutions	Increse staining time
Fatigued(old) stains	Change stains
Another slide coverd specimen during staining	Keep slides separate
Uneven Staining
Variation of pH in different areas of slide surface	Use new slides and avoid touching their surface before and after preparation
Water allowed to stand on some areas of the slide after staining and washing	Tilt slides close to vertical to drain water from the surface or dry with a fan
Inadequate mixing of stain and buffer	Mix stain and buffer thoroughly
Precipitate on Preparation
Inadequate stain filtration	Filter or change the stains
Inadequate washing of slide after staining	Rinse slides well after staining
Dirty slides used	Use clean new slides
Stain solution dries during staining	Use sufficient stain and do not leave it on slide too long
Miscellaneous
Overstained preparation	Destain with 95% methanol and restain
Refractile artifact on RBC with Diff-Quik stain	Change the fixative

*Reference: Cowell & Tyler (1992), Cytology and Haematology of the Horse.

Cheers
Douglas

SIP

Subject Title: Clinical Chemistry
Name of Test: Total Carotene Test


Carotene is an orange photosynthetic pigment important for photosynthesis. The characteristic orange colour of carrots and red yellow pigments in fruits and vegetables are due to carotenoids. It comes in two primary forms; alpha-carotene and beta-carotene. Beta-carotene is probably the best known of them and is a precursor of Vitamin A. Carotene is stored in the liver and converted to Vitamin A as needed. In addition, carotene is an antioxidant nutrient so it can reduce the risks of cardiovascular diseases and some forms of cancer.


Principle and Procedure of Test
In this test, protein are first removed by precipitation with ethanol. The carotene and other form of carotenoids are extracted with petroleum ether. The characteristics absorbance is measured at 450nm.

Before running the patient sample, two levels of controls are run. Calculations are done to obtain the concentration of carotene. After ensuring that the QC results are acceptable, we will run the patient sample.
1) Pipette 1mL of patient serum sample into the glass test-tube

2) Add 1ml of absolute ethanol into the glass test-tube

3) Vortex the glass test-tube

4) Add 2ml of petroleum ether and vortex them for 10 min to ensure they are well mixed

5) Allow the glass test-tube to stand for 5min

6) After 5 min, extract the top layer of ether and transfer them into the glass microcurvette until it is filled to the top for absorbance readings. This is to enable complete light transmission through the solution

7) Using petroleum ether as blank, place the glass microcurvette into the spectrometer. Check for turbidity in the sample as it can cause false high absorbance readings

8) The absorbance is measured at 450nm

9) Calculations are done to obtain the concentration of carotene. This is as follow:

ODdiff X 14.59 = ____µmol/L

*Note: ODdiff = OD (450nm) – OD blank


Reference Range: 0.93 to 3.73µmol/L


Clinical Interpretation
Concentrations above the reference range shows a high levels of carotene in the body known as carotenemia. Unlike excess Vitamin A, excess carotene is non-toxic. Although carotenemia is not dangerous, it can lead to yellowish/orange discolouration of the skin, mostly occurring in the palms of hands and the soles of the feet. This is due to the consumption of abundance carotene from foods (such as carrot juice) or from supplements. It is nearly impossible to overdose on carotene because the body excretes the excess. This can also be a medical sign of other conditions such as renal diseases. Since carotene itself is harmless, it does not require treatment. Reducing high quantities intake of carotene will gradually remove the colourisation of skin. Sometimes yellowish discolouration of the skin can be misdiagnosed as jaundice. Jaundice affects the colour of the eye while carotenemia does not. A bilirubin serum test can be used to identify whether jaundice is involved.

Concentrations below the reference range shows there is carotene deficiency. Such deficiencies are seldom seen, however, even in people who don't eat fruits or vegetables or take supplements, because so many other foods supply the nutrient. Symptoms of carotene deficiency include dry skin, night blindness and susceptibility to infection.

Total carotene test is a valuable part of diagnosis of Vitamin A deficiency or excessive Vitamin A. This is because carotene is converted to Vitamin A in the body. However, the actual diagnosis requires interpretation of test result in conjunction with other clinical findings.


Soong Ci Liang
0503333G
TG01

CELL-DYN 3700: Principles of Operation

Sample Aspiration

There is 2 modes of aspiration.

• Open Sampler Mode is used to aspirate sample from a collection tube that has been opened and is held under the Open Sample Aspiration Probe.
• Manual Closed Sampler Mode or Automated Sample LoaderMode aspirate blood directly from a capped collection tube by piercing the tube stopper.

Aspiration volume for open mode is 130uL ± 5%, 240uL ± 5% for close mode, and 355uL ± 5% for Sample Loader mode. Sample is aspirated into the Analyzer by the Aspiration Peristaltic Pump, through the Shear Valve.

White Blood Cells(WBC) Analysis

2 measurements: WBC optical count (WOC) and WBC impedance count (WIC).

1. WOC Sheath Syringe dispenses 1.6mL of Sheath Reagent through the Shear Valve, picking up 32uL sample.
2. Sample segment and sheath are then routed to WOC mixing chamber where dilution is bubble-mixed. (1:51)
3. WOC Peristaltic Pump transfers the WOC dilution from the WOC Mixing Chamber to Sample Feed Nozzle in WOC flow cell.
4. A stream of WOC Sheath Reagent is directed through the Flow Cell.
5. WOC Metering Syringe injects 78uL of WOC dilution into the Flow Cellsheath stream.
6. A laser beam is focused on the Flow Cell. As the sample stream intersects the laser beam, the light scattered by the cells is measured at four different angular intervals.

1. WIC/HGB Diluent Syringe dispense 5.25mL of Diluent through Shear Valve, picking up the 20uL WIC/HGB sample segment.
2. Segment and Diluent fouted to Mixing Chamber in the von Beherns WIC Transducer. At the same time, WIC/HGB Lyse Syringe delivers 0.75mL of WIC/HGB Lyse to mixing chamber.
3. The dilution is bubble-mixed. Final dilution is 1:301.
4. Dilution is pulled through the aperture by vacuum, Volumetric metering ensures 200uL used for measurement.
5. Electrical Impedance is used to count WBC as the traverse the aperture.
6. When the count portion of the cycle is completed, the aperture is auto-cleaned by Aperture cleaning circuit.

RBC/PLT Analysis

1. RBC Diluent Syringe dispenses 7.2mL diluent through Shear Valve, picking up 0.74uL RBC/PLT sample segment
2. Sample segment and diluent routed to mixing chamber of von Behrens RBC/PLT Transducer where dilution is bubble-mixed. (1:9760)
3. Dilution is pulled through the aperture by vacuum. Volumetric metering ensures that 100uL of dilution is measured.
4. Electrical Impedance is used to count RBCs and PLTs as they traverse the aperture.

Haemoglobin Analysis

1. After 200uL of WIC /HBG are metered through WIC aperture, remaining dilution is transferred to HGB Flow Cell.
2. HGB concentration is measured spectrophotometrically.

*All information obatined from ABBOTT CELL-DYN 3700 operator's manual.

Cheers
Douglas