Mohd Farid Laporan Latihan Industri Sarjana Muda Sains Bioperubatan UKM Tahun 3 Sesi 2007/2008-Hospital Selayang Part VIII-Haematology And Blood Tranfusion Unit

Haematology And Blood Transfusion Unit

ROUTINE WORK DONE IN HEMATOLOGY LABORATORY

1. FULL BLOOD COUNT

Principle

SE 9000 Analyzer is an automated full blood counter machine. This machine can make measurement of hemoglobin concentration, measurement relating to red cells and platelets counting and differential counting of leucocytes.

SE 9000 Hematology Analyzer:

Introduction

a) System Overview

The SE 9000 Analyzer consists of five major systems:

1. The main unit

Houses of the hydraulic and electronic system for analysis and their control component

2. Sampler unit

Consist of a mixer, cup piercer unit which supply samples to the main unit

3. Data management System (DMS)

Processes and display data obtain from the main unit

4. Pneumatic unit

Supplies the required pressure and vacuum for analysis

5. Power supply

Supplies power to the main unit

b) Sample Process

Analysis Parameter

The SE9000 uses 5 detection methods and 8 types of reagent to analyze the 23 parameters as follows.

Parameter	Code
White blood cell count	WBC
Red blood cell count	RBC
Hemoglobin concentration	HGB
Hematocrit	HCT
Mean Corpuscular Volume	MCV
Mean Corpuscular Hemoglobin	MCH
Mean Corpuscular Hemoglobin Concentration	MCHC
Platelet count	PLT
Neutrophil Percent	NEUT%
Lymphocyte Percent	LYMPH%
Monocyte Percent	MONO%
Eosinophil Percent	EO%
Basophil Percent	BASO%
Neutrophil Percent	NEUT#
Lymphocyte Percent	LYMPH#
Monocyte Count	MONO#
Eosinophil Count	EO#
Basophil Count	BASO#
RBC Distribution Width	RDW-SD
RBC Distribution Width	RDW-CV
Platelet Distribution Width	PDW
Mean Platelet Volume	MPV
Platelet Large Cell Ratio	P-LCR

Reagent For SE9000

CELL PACK

CELL PACK 3D (II)

STROMATOLYSER 3D (II)

STROMATOLYSER-BA

STROMATOLYSER-EO (II)

STROMATOLYSER-IM

CELL SHEALTH

SULFOLYSEZ

MONORESH

Diluted CELL CLEAN

Normal range

Full Blood Count

	Neo	child	Male (Adult)	Female (adult)
Hb	10-23	10-15	13-17	12-16
RBC	4.8-7.0	3.8-5.4	4.2-6.0	3.8-5.4
PCV	50-70	32-44	38-50	36-46
MCV	95-120	75-88	85-95 27-39 32-36 150-400 4-11
MCH	36-40	25-29
MCHC	30-34	31-35
Plt	100-350	150-400
WBC	10-25	5-15

Differential Count

	Neo	Child	Male (adult)	Female( adult)
Neutrophil	40-75	35-65	45-70 20-45 0-5 0-2 0.5-1.5 0-20
Lymphocyte	20-40	25-75
Monocyte	2-10	3-9
Eosinophil	0-5	0-5
Basophil	0-2	0-2
Reticulocyte	1-5	0.5-1.5
ESR		0-10

Method

• Receive blood sample in a tube with purple cover which containing patient blood mix with EDTA
• Log in patient acc no in computer under hematology lab
• Put sample in the rack
• Set the rack on the sampler. Make sure label is in proper location to allow analyzer to scan the barcode of each sample
• Press [SAMPLER START/STOP] key to start analysis.
• The instrument performs mixing, aspirating and analyzing automatically. After analysis, the results are displayed on the computer screen and will automatically transferred to the result data in the hospital computer system.
• For children sample, analysis was done manually by entering patient acc no and hold patient sample on the straw and press start. Result will automatically be transferred into computer system.
• Result from analyzer will be verify by the in charge MLT

Results

• Normally interpretation will focus on white blood cell, hemoglobin and platelet cell.
• If white blood cell low than normal, suspected virus infection
• If white blood cell high than normal, suspected bacteria infection
• If white blood cell double than normal suspected leukemia
• If hemoglobin low is suspected internal bleeding
• If platelet less than 150x10^-9 /L is suspected dengue
• If blood is not mixed totally and there is blood clotting sample will be rejected because it can cause false interpretation of result.

2. FULL BLOOD PICTURE (FBP)

Principle

Full blood picture done because some of disease cannot see through the quantity of the full blood count but it must observe by the structure of the cells. SE Alpha is the fully automated machine that run the reticulocyte count and made full blood picture slide. The slide blood film is stained by Leishman’s Stain using the SE Alpha.

Method

Put the blood into the plain test tube

Enter patient acc no by reading the printed barcode manually

Press start button to run the machine

Machine will suck automatically and analyzed it.

The result will be sent to host computer and printed

Full blood picture slide is prepared

Result

Nuclei	Color
Cytoplasm:
Erythrocytes	Deep pink
Reticulocytes	Grey blue
Neutrophils	Orange pink
Lymphocytes	Blue, some small lymphocytes deep blue
Monocytes	Grey-blue
Basophils	Blue
Granules:
Neutrophils	Fine purple
Eosinophils	Red orange
Basophils	Purple black
Monocytes	Fine reddish
Platelets	Purple

3. ERYTHROCYTE SEDIMENTATION RATE (ESR)

Principle

• If blood to which an anticoagulant Trisodium citrate3.8% has been added is allowed to stand in a tube, the cell will settle down to the bottom, leaving the plasma as a clear supernatant fluid, the rate at which the cells settle is known as the Erythrocyte Sedimentation Rate (ESR).
• The ESR measures the distance red blood cells will fall along the length of a vertical tube over a given time period. A raised ESR reflects an increased production of acute phase proteins. Although it is a non-specific phenomenon, it is clinically useful in certain chronic disorders, e.g. rheumatoid arthritis or tuberculosis, as an index of progress of the disease
• A normal ESR does not exclude organic disease but, on the other hand, the vast majority of acute or chronic infection and most neoplastic and degenerative disease are associated with changes in the plasma proteins which lead to an acceleration in sedimentation.
• The blood specimen is collected in Westergen Sedimentation Tube. The machine used to analyze ESR is ELECTA Model Monitor-S.
• Normal range: 0-20 mm/hr

Method

• Put the blood into the Westergen sedimentation tube which contain Trisodium citrate3.8%
• Mix sample well
• Key in patient acc no into the machine, place tube into the machine according to place that telah ditentukan by the machine
• Leave undisturbed for 60 minutes.
• After finish analyzing, result will be printed out automatically

Interpretation

• Normal RBC is destroying about 120 days. If the RBC is destroyed before 120 days, because of not enough rest, less vitamin B or not enough nutrients. This will cause ESR result higher from the normal range
• As for female, if the ESR more than 50mm/hour with the other things is normal, doctor will suspect other disease

4. PROTROMBIN TIME (PT)

Principle:

STA Compact

Introduction

STA Compact^® is an automated laboratory instrument designed to perform invitro test which aid in the diagnosis of coagulation abnormalities as well as to assist in monitoring anti coagulant therapy. The instrument is capable of performing clotting assays as well as chromogenic and immunological assays on plasma samples. Prothrombin time activated prothrombin time and fibrinogen was calculated using this instrument.

Principle

Clotting Method

• The detection system for clotting time assay is on the STA Compact^® is based on the increase of viscosity of the plasma being tested. This increase of viscosity is measured through the motion of a sickness steel ball that is made to effect pendular swings on the two curved rail track provided in the bottom of the cuvette containing the test plasma.
• Constant pendular swings of the ball are created by electromagnetic field that is applied alternately on opposite sides of the cuvette by two independent coils. The energy of the electromagnetic field can be varied depending on the test being performed (weak clot for fibrinogen, normal clot for other)
• At constant plasma viscosity, ball motion remains constant. However, as soon as the plasma started to clot, (as a result of the coagulation process being initiated by the addition of the clot starting reagent), the viscosity of the plasma starts to increase, and their change in plasma viscosity affects ball movement, slowing it down. As the viscosity increases, the oscillation amplitude of the ball swing decreases.

Photometric Method

• The detection of chromogenic assay on STA Compact^® is based on the absorbance (optical density) of monochromatic (405nm or 540 nm) light passing through the cuvette as a chromogenic reaction take place.
• Incident light (Io) entering the cuvette is partially absorbed by the reaction mixture as it passes through. The transmitted light is measured (I+Ip), and converted to absorbance by following equation
• A:-log (I1/IO)
• The effect stray light is eliminated by taking two fairly close measurement of the light transmitted

• I1 = I+Ip (first incident which include incident light and stray light)
• I2 = Ip (second measurement while blocking the incident light, correspond to the stray light)

• When I2 is subtracted from I1, the result is I2 which is only the light transmitted from the incident light. Ip is assumed to remain constant between the two measurements. Incident light is provided by a tungsten-halogen lamp and is made monochromatic by passing through a 405nm, or 540nm, interference filter. This step occurs inside the optical module. A system of fiber optic carries the monochromatic light from the optical to the measurement head. Another set of optical fibers carries the transmitted light from the measurement head to the photometry measurement board.

Method

• The primary patient sample tubes and the dilution buffers are loaded in the sample drawer. The position of each sample is automatically detected by the identification system
• The control plasma vials, the calibration plasma vials as well as the reagent vials are loaded in the product drawer where the temperature is monitored between 15⁰C and 19⁰Cby a system based on Peltier elements. The position of each vial is also automatically detected by the positive identification system.
• Sample plasma, control plasmas as well as calibrator plasmas are pipetted by needle no 1 (pipetting head)

PT	APTT	FIB	Possible conditions
N	N	N	Normal haemostasis
			Thrombocytopenia*
			Disorder of platelet function
			Vascular disorder
			Bleeding from severely damaged vessel
			Very mild FactorVIII deficiency
Long	N	N	Factor VII deficiency (rare)
			A start of oral anticoagulant therapy
			Factor VII, IX deficiency
			Factor XI, XII deficiency (rare)
			Von Willebrand disease
			Circulating anticoagulant
			Prekallikrein or kininogen deficiency (rare)
Long	Long	N	Vitamin K deficiency
			Oral anticoagulant
			Factor II, V, X deficiency (rare)
			Liver disease*
			Massive blood transfusion*
Long	Long	Long	Heparin
			Fibrinogen deficiency
			Hyperfibrinolysis
			DIC*
			Acute liver disease*

Normal *Thrombocytopenia present

Factor II = protrombin

Factor VIII deficiency =Hemophilia A

Factor IX deficiency = Hemophilia B

5. G-6-PD DEFICIENCY SCREENING TEST

Principle

The test is to screen for G-6-PD deficiency in newborn. It is a screening test for dried blood spot and whole blood samples.

The production of NADPH will produce fluorescent under UV-Light. If there is a marked deficiency G-6-PD, no fluorescent will be observed

Method

• A disk of blood stain paper of 5mm diameter is punched out.
• Introduce into a vial-1 blood paper disk & Reagent solution
• Mix well; incubate for 10 minutes under room temperature.
• Apply 0.01 µl of the mixture to a filter paper and allow it to dry for several minutes.
• View the fluorescent under long wave UV-light in a dark surrounding

Results

Samples obtained from normal or slightly reduced G-6-PDH activity will show strong fluorescent under the UV-light. Failure to fluoresce after 10 minutes incubation suggest total marked deficiency of G-6-PDH

6. LE CELL TEST

LE Cell test is a qualitative determination of anti-DNP associated with Systemic Lupus Erythematosis in human serum. This test used latex agglutination method. Reagent that been used in this test is PLASMATEC s-LE latex test. In s-LE, autoantibodies directed against native deoxyribonucleic acid and other nuclear constituents are produced. It is classed as the prototype of severe autoimmune diseases, involving a variety of tissues and associated with a wide range of antibodies in the circulation. 

Characteristic of the disease are antibodies against native DNA, nucleoprotein, denaturated DNA and other extractable nuclear antigens. S-LE also affects a wide range of tissue. Organ affected are in decreasing incidence, joints, skin, kidney, central nervous system, heart and lungs. One other important feature is the high frequency of the disease in women, approximately 3to 4 times more frequent than in men.

In this test, latex particle of reagent will bound with native deoxyribonucleic protein (DNP) by means of an intermediary albumin matrix. This coated latex particle combine with anti-DNP antibodies in serum to give a visible agglutination. Visible agglutination at any degree is considered as positive s-LE.

7. BONE MARROW ASPIRATION AND TREPHINE BIOPSY

ROUTINE WORK IN BLOOD BANK

Blood Supply

In Selayang hospital, blood component was ordered form Pusat Darah Negara. Blood component that been taken from Pusat Darah Negara will be log in into blood bank computer system. After each product been log in, it will be kept in the blood bank refrigerator depend on type of the blood component until it reaches its expiry date.

Tests

a. Blood grouping

I. ABO forward grouping

Introduction

It is a test to determine patient blood group; either A, B, AB or O.It is important for patient that need blood transfusion. This test is to identify unknown antigen of patient red blood cells using known antiserum. Each antiserum contains one type of antibody, which react specifically with antigen in patient’s blood. The antiserum is manufactured from human serum. These antisera are summarized in table below

Antiserum	Color	Source
Anti-A	Blue	Group B donor
Anti-B	Yellow	Group A donor

Procedure

1 drop of each antiserum is delivered on different test tube

1 drop of patient whole blood is added and mixed well in the test tube

The presence of agglutination in each test tube is observed.

Results

Blood Group	Anti-A	Anti-B
A	+	-
B	-	+
AB	+	+
O	-	-

(+)-Agglutination

(-)-No agglutination

II. ABO Reverse Grouping

Introduction

This test uses patient’s serum containing unknown antibodies and is tested with cell that having known antigen

Procedure

• 1 drop of each antigen is delivered on different test tube
• 1 drop of patient serum is added and mixed well in the test tube
• The presence of agglutination in each test tube is observed.

Results

Blood group	Antigen A	Antigen B	Antigen O
A	-	+	-
B	+	-	-
AB	-	-	-
O	+	+	-

(+)-Agglutination

(-)-No Agglutination

III. Rhesus

Apart of ABO grouping, Rhesus grouping must also been done. Rhesus grouping is tested using anti-Rh or anti-D. If patient blood was screened as Rh negative, therefore the blood must be further test for weak-D or Duffy. If both test give negative result, therefore it is confirmed as Rh negative.

Procedure

• 1 drop of Anti-D is delivered on a test tube
• 1 drop of patient whole blood is added and mixed well in the test tube
• The presence of agglutination in each test tube is observed.

Results

Rhesus	Anti-D/anti-Rh
Rhesus positive	Agglutination
Rhesus negative	No agglutination

Rh D Phenotyping

Rh D phenotyping is a further test for confirmation of rhesus negative patient. Rh phenotyping was carried out using ID card RhD phenotype

Principle

The expression Rh positive or Rh negative is based on the presence or absence of the Rh D antigen on the red cells. To detect the presence or absence of the Rh D antigen on the red cells, anti-D serum is used which can be of human or monoclonal origin. Patient whole blood sample will be diluted to 5% red blood suspension in ID-Diluent and incubated for 10 minute.10 ul of suspension was piped into each microtube ant will be centrifuged for 10 minutes.

Results

• Positive: Agglutination cell forming a red line on surface of the gel or agglutination dispensed in the gel.
• Negative: Compact button of cells on the bottom of the microtube 

b. Antibody screening

This procedure applies to test the antibody screening including pre-transfusion and prenatal testing. The screening for unexpected antibodies has been aim of detecting the clinically significant antibodies present in the patient’s sample. The autocontrol will indicate, in positive screening of unexpected antibodies, whether it is due to the presence of antibody, an alloantibody or both. This test is carried out using S.A gel card, Serascan Diana 2 reagent and Serascn Diana 2P reagent

Principle

An antibody reacts specifically with the antigen which stimulated its production. According to this, an antibody can be identified depending on its pattern of reactivity when confronted with a panel of reagent red blood cell with a known antigenic configuration.

The principal of the test is based on the gel technique for detection of red cell agglutination reactions. The agglutination occurs when the red cell antigens contact the corresponding antibodies, present in the reagent or in the serum or plasma sample.

Result

• Positive: Agglutination cell forming a red line on surface of the gel or agglutination dispensed in the gel. Positive for presence of antibody
• Negative: Compact button of cells on the bottom of the microtube .Absence of antibody 

c. Crossmatching

Introduction

It is a test of the compatibility of donor and recipient blood performed before transfusion. Red cells of the donor are placed in serum of the recipient (major crossmatch) and red cells of the recipient in serum donor (minor crossmatch) and antiglobulin is added to increase reactivity; the presence of hemolysis agglutination indicates incompatibility. Using LISS/Coombs.

Principle Of Test Method

The crossmatch test is detecting the presence of antibodies in the recipient’s serum, including anti-A and anti-B that could destroy transfused red cells. A positive result in the crossmatch test requires explanation and the patient should not receive a transfusion until the cause of incompatibility has been fully determined. The objective of testing is to select donor units that are able to provide maximal benefit to the patient.

After screening antibody, procedure proceeds to major crossmatch. After patient antibody screening test negative in alloantibody red blood cell, patient blood and potential donor blood was been crossmatch. Crossmatch test is carried out to detect the presence of antibodies in the recipient’s serum, including anti-A and anti-B that could destroy transfused red cells. A positive result in the crossmatch test requires explanation, and the patient should not receive transfusion until the cause of incompatibility has bees fully determined. The objective of incompatibility is to select donor unit that are able to provide maximal benefit to the patient.

Here in Selayang Hospital, crossmatch test is carried out using ID card LISS/Coombs

Clinical Significant

Polyspecific anti-human globulin (AHG) reagents are used for routine alloantibody detection and identification, compatibility tests and the direct antiglobulin test (DAT).

The most important function of the polyspecific AGH reagent is to detect the presence of the IgG. The importances of anticomplement in the AGH reagent debatable since antibody detectable only by their ability to bind complement are rather rare. However, anti c3d activity is important for the DAT in the investigation of autoimmune hemolytic anemia. A positive DAT generally indicates that the red cells are coated in vivo with immunoglobulin and/or complement.

The microtube of ID-card LISS/ Coombs contain polyspecific AHG to be used for antibody screening, antibody identification, cross match and DAT. For the indirect antiglobulin test (IAT), labour intensive washing procedure are eliminated, due to the fact that red cell suspension is added to the microtube before the plasma, creating a barrier over the gel suspension, thus avoiding neutralization of the AHG by serum IgG protein.

The ID card LISS/ Coombs is suitable for DAT, for the compatibility test, for antibody screening and identification.

The most important function of the polyspecific AGH reagent is to detect the presence of IgG. The importance of anticomplement in the AGH reagent is debatable since antibody detectable only by its ability to bind complement is rather rare. However

Procedure

• Carry out ABO grouping and Rh test
• Choose donor blood that is in same group with patient blood.
• Spin patient sample. Separate patient serum
• Label patient ID test LISS/Coombs card with patient accession number, remove aluminium foil
• Label blood bag and cut segment from blood bag to prepare 0.8% red cell suspension. Pipette 50ul of each 0.8% cell suspension into microtubes
• Pipette 25ul of patient serum into each microtube
• Incubate at 37oC for 15 minutes
• Centrifuge in ID-centrifuge at 3000 rpm for 10 minutes
• Interpreted result
• Verify result in computer
• Place blood tag on the blood units and place it in crossmatch blood fridge.

Definition/Result

• Positive: Agglutination cell forming a red line on surface of the gel or agglutination dispensed in the gel. Donor blood is not compatible to patient
• Negative: Compact button of cells on the bottom of the microtube. Donor blood is compatible to patient.

d. Direct Coombs Test

Introduction

A test for the presence of non-agglutinating antibodies against red cell that uses anti human globulin antibody to agglutinate red cells coated with the non-agglutinating antibody. It detects antibodies bound to circulating red cells in vivo. It is used in the evaluation of autoimmune and drug induced hemolytic anemia and hemolytic disease of the newborn.

Releasing Blood And Disposal Of The Transfused Bags

Blood that have been crossmatch will only be being released to ward if there is request from the doctor.