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INTRODUCTION TO PRACTICAL HAEMATOLOGY

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  • INTRODUCTION TO PRACTICAL HAEMATOLOGY

    INTRODUCTION TO PRACTICAL HAEMATOLOGY

    Introduction:-

    In the past humankind know about presence of blood, but a few centuries back science discovered that this blood circulates in our body. In Greek, Roman, and Unani medicine describe some disease due to blood abnormalities into: traumatic, inflammatory, infective, and neoplastic.


    DEFINATION OF BLOOD:

    Blood is highly specialized (sterile) connective tissues, which circulate in a closed system of vessels as a liquid with red coluor, but in out this system a solid phase will perform, which we called plug or blood clot.

    Haematology: is the science that study the blood, and it's structure, function, disease, and the convenience between structure and the function.

    BLOOD COMPONENT

    Mainly we can divide blood into two parts:
    1) Plasma 2) Blood cells.

    The total amount of blood approximately 1/14 of the total body weight or 60-70 ml/ each kilogram of body weight.
    Blood flows through every organ of the body providing effective communication between tissues.

    PLASMA:
    Plasma is a pale yellow fluid in which blood cells are suspended in.
    Plasma forms about 55% of blood volume and composed of 95%or more water, and many solutes including proteins, minerals, ions, organic materials, hormones, enzymes, products of digestion, and waste products.



    BLOOD CELLS:
    1) Red blood cells (RBC).
    2) White blood cells (WBC).
    3) Platelets.

    FUNCTION OF BLOOD:

    □ Transportation and distribution:
    - Oxygen transportation by haemoglobin from lungs to the tissues.
    - Blood also can transport the nutrients absorbed by the digestive system to the tissues for use or storage.
    - Hormones are carried from endocrine glands to the organs.
    - Wastes are transported from tissues for excretion e.g.: carbon , dioxide, urea, createnine,…

    □ Regulatory:
    - Plasma maintain the PH. of blood (7.35-7.45), and in the tissues .
    - Osmotic pressure in plasma is regulate by proteins and salts (sodium, chloride) to prevent excessive loss of fluids from the blood into tissues.
    - Regulation of the body temperature.

    □ Protective:
    - Platelets and coagulation factors control the blood loss by thrombous formation.
    - Leukocytes defend and produce antibodies and toxin against infection and tumor cells.




    HAEMATOPOIESIS:
    In normal healthy person there is a constant break down and new formation of cells, and the procedure of blood cells formation called Haematopoiesis.


    NORMAL SITES OF BLOOD FORMATION:
    - Fetus:
    * Less than 2 months: in Yolk sac.
    * 2-7 months: in the liver and a few in the spleen.
    * Full term: in bone marrow for RBC, PLTs, and granulocytes, but lymphocytes and monocytes occures in spleen, lymph nodes and lymphoid tissues (liver and bone marrow with less numbers).


    - After birth:
    Mainly from bone marrow even monocytes, except lymphocytes still from spleen and lymph tissues.



    - In adult:
    Main sites of haematopoiesis are the vertebrae, ribs, sternum, skull bones, pelvis, sacrum, and proximal ends of femur and humerus.
    Haematopoiesis can be sub-divided into 3 stages:
    1) Mesoblastic period.
    2) Hepatic period.
    3) Myeloid period.

    ABNORMAL SITES OF HAEMATOPOIESIS:
    In certain disorders the fetal haematopoitic organs revert to their old function supported by the reticulum cells, this occurs when bone marrow can not fulfill the requirements or demand for new cells, this called EXTRA-MEDALLARY haematopoiesis, (Myeloid metaplasia).
    In some rare cases adrenal glands, cartilages, adipose tissues, intra thoracic areas, kidneys, and endo-sternum can produce blood cells.



    PRE-ANALYSIS MANAGEMENT

    The clinical laboratory is useful to assist in diagnosis, and management of patient.

    A test request is a request for consultative services, to generate a laboratory report using to make clinical judgments.
    - Understanding of the test process and procedures, collection, and handling enables the laboratory staff to achieve more nearly optimal conditions and consequently to improve the accuracy and precision of each measurement.


    REASONS FOR ORDERING LAB. REQUEST:
    1) To confirm a clinical impression or diagnosis.
    2) To rule out a diagnosis.
    3) To monitor therapy.
    4) To establish prognosis.
    5) To screen for or detect disease.






    TEST REQUESTATION:

    The physician initiates the test request by writing an order for lab. examinations in the patient medical chart/record.
    - The orders are carry out to an appropriate lab. request form by the nursing station or secretary unit.
    - Each laboratory form has a list of test with reference intervals and a space for the result.
    - Patient data (demographics) include, patient name, sex, age, date of admission, date of test ordering, room number,……..must be clearly writing on the request, or patient's addressograph plate or computerized label are stamped onto the request.
    - The requests are given to the collect unit or to the phlebotomist /nurse to draw the specimen, specimen tube must be labeled be for the specimen is drawn.
    ☼ It is essential to follow strict quality control procedures through all stages of test request to avoid several possible errors, such as, incorrect or missing test entering, ……
    - All requests should have a full, clear patient data, and correct sample labeling.
    - Samples quantity and anticoagulant should be suitable, and with no haemolysis, or clot.






    BLOOD SPECIMEN COLLECTION:


    A. SKIN PUNCTURE:
    Skin puncture is the method of choice in pediatric patients, especially infants.
    - Skin puncture can be used in adults with:
    * extreme obesity.
    * sever burn.
    * thrombotic tendencies.
    Technique:
    1) Select an appropriate puncture site, lateral or medial plantar heel surface for infants, in older infants the palmer surface of the last digit of the second, third, or forth fingers (big toe, and ear lobe can be used).
    -The site of puncture must not be edematous or a previous puncture site.
    2) Warm the puncture site with a warm moist towel, and clean the puncture site with 70%of aqueous isopropanol solution, allow the area to dray.
    3) Make the puncture with a sterile lancet with blade no longer than 2.4 mm.
    4) Discard the first drop of blood by wiping it away with a sterile pad.
    5) Collect the specimen in a suitable container (oral aspiration of blood is discouraged for a safety reasons ).
    5) Label the specimen container.



    B. VENOUS PUNCTURE:
    Technique:
    1) Identify the patient by checking identification card against the request, ask the conscious patient his/her full name and birth date (do not draw any specimen without properly identification).
    2) If fasting specimen is required , confirm that the fasting order has been followed.
    3) Inform the patient, what is to be done and reassure the patient to avoid as much tension as possible.
    4) Position the patient properly for easy, comfortable access to the antecubital fossa.
    5) Assemble equipments and supply (tubes, tourniquet, syringes…..).
    6) Ask the patient to make a fist –to make the veins more palpable – then select a suitable vein (veins of atecubital fossa , in particular the median and cephalic veins are preferred), wrist , ankle, and hands veins may also be used.
    If one arm has an intravenous line , use the other arm to draw a blood sample.
    7) Clean the venipuncture site with 70%isopropyl alcohol solution in a circular motion and allow the area to dray.
    8) Apply a tourniquet above the puncture site, "never leave the tourniquet longer than 1min.
    9) Use your thumb and middle finger or thumb and the index finger, to anchor the vein.
    10) Enter the skin with the bevel of the needle at 15 degree angle, to the arm with the arm, with the bevel up , insert the needle smoothly, and fairly fast.
    - If using a syringe pull pack on the barrel with a slow until the blood flows into the syringe (do not pull back too quickly to avoid the haemolysis or collapsing the vein).
    - If using a vacutainer, as soon as the needle is in the vein ease the tube fore ward in the holder, at the same time hold the needle firmly in place.
    11) At the end of the collection release the tourniquet.
    12) After all blood samples have been drawn, have the patient relax his fist.
    13) Place a clean, sterile, dray cotton ball over the site and withdraw the needle , and apply a pressure to the site , and bandage the arm.
    14) Mix the blood with the anticoagulant.
    15) Check the patient condition e.g.: whither he is faint, bleeding is under control,…..
    16) Dispose the contaminated materials.

    C. ARTERIAL PUNCTURE:
    Arterial blood is used to measure oxygen, carbon dioxide , and measuring PH.,…
    Arterial punctures are technically more difficult to perform.
    Technique:
    1) Select the puncture site, the radial artery is the most common site, if the ulnar artery is absent, do not puncture the radial artery (use the Allen test to make sure of collateral circulation ).
    The femoral artery and the brachial artery, at the antecubital fossa provide alternative sites for puncture, scalp arteries are used in infant.
    2) Anesthetized the puncture site if necessary, prepare the syringe by aspirate an anticoagulant (usually heparin).
    3) Record the patient temperature and perform Allen test (for radial artery puncture) as follow:
    - compress the radial and ulnar arteries at the wrist until the palm of the hand becomes blanched.
    - Release the pressure from ulnar artery and observe that the hand becomes flushed, if the hand remains blanched DO NOT puncture the radial artery.
    4) Clean the site, place a finger over the artery and puncture the skin 5-10mm. distal to the finger .
    - Blood rushing into the needle, or pull back on the plunger and obtain the required amount of the blood.
    5) Quickly withdraw the needle and syringe , place a sterile cotton ball or dry gauze over the puncture site.
    6) Apply firm pressure for at least 5 min.
    7) Expel any air bubble from the syringe.
    8) Remove the needle and cap it with a tight – fitting "Luer cap" and mix the anticoagulant by gentile inversing of the syringe.
    9) Label the sample and place it in an ice/ice water bath.
    10) Transport the sample on ice immediately to the lab.
    * Drawing problems:

    Occasionally, the phlebotomists are unable to obtain blood by ordinary venipuncture.
    - In some cases a skin puncture may suffice; if not, a physician draws the specimen using most commonly the femoral vain or jugular vain in children.








    REAGENTS:

    Reagent contain chemicals exist in varying degrees of purity. – all reagent should have a label identify the concentration , purity, amount, and compounds,….
    - Reagents or chemicals arrived into lab. with a certain guarantee of purity ;once the seal is broken, the guaranteed analysis is strictly in the hands of the receiving lab.
    - Definite steps must be taken to ensure that the reagent/ chemicals are handled under optimal condition.
    - It extremely important to read the label for proper storage .
    - Never sample directly from the reagent bottle.
    - It essential that each lab. first evaluate a kit / reagent according to an established protocol, then monitor kit/reagent performance by appropriate quality control procedures.
    - In generally distilled water and deionized water most commonly used to prepare reagents.
    - The College of American Pathologist has drawn up specification and methods of quality control for reagent water.
    * Three grades of water are defined:
    ▫ Type I reagent water:
    For procedures which require maximum water purity
    - Preparation of standard solutions
    - Ultra micro-chemical analysis.
    - Measurement of nanogram or sub nanogram concentration.
    ▫ Type II reagent water:
    For most lab. testing, in chemistry, haematology, immunology, and other clinical tests.
    ▫ Type III reagent water:
    For most qualitative testing; most procedure in urinalysis, parasitology and for washing glass ware.
    * Carbon dioxide free water is used in gasses such as Co2 , ammonia and O2 may affect analysis.



    SOME METHODOLGY PRINCIBLS USED IN CLINICAL LABORATORY



    □ PHOTOMERTRY:
    Photometric measurement defined as measurement of light intensity of multiple wave length.
    SPECTROPHOTOMETRY: meant measurement of light intensity in a much narrower wave length range.

    □ FLAME PHOTOMETRY:
    Heat energy of a flame makes the electrons in an atom excited and being unstable, then the electrons give up their excess energy to the environment as they change from the higher energy state (level) to a lower energy state as light, the light may consist of one or more than one energy level, therefore many different wave lengths; these wave lengths are individually characteristic for each element.

    □ FLUOROMERTRY:
    Fluorescence is a physical energy process that occurs when certain compounds absorb electromagnetic radiation and become excited, and then return to energy level slightly higher than or equal to their original energy level , the energy given off is less than or equal to that absorbed, and wave length will be longer or equal to that absorbed for excitation.

    □ TURBIDIMETRY:
    Turbidimetry measures the amount of light blocked by particulate matter as light passes through the cuvette.


    □ POTENTIOMETRY:
    The measurement of the potential voltage between two electrodes in solution from the basis for a variety of measurement that can be used to quantitate concentration of substance of interest.

    □ ION SELECTIVE ELECTRODES (ISE):
    There are three different basic ISE. Classes:
    - Ion selective glass.
    - Solid state electrode.
    - Liquid ion exchange membranes.

    □ CHROMATOGRAPHY:
    The purpose of chromatography involves separation of a mixture on the basis of specific difference of the physical – chemical characteristics of the components.




    * Steps involved in requesting, performing, and evaluating measured quantity :


    (I) Physician request a quantitative measurement of a constituent in biological specimen.
    (II) Laboratory personnel perform the assay:
    A. Pre-instrumental phase
    1- Preparation of the patient.
    2- Obtain the specimen
    3- Processing the specimen
    4- Storing the specimen prior the measuring steps.
    B. Instrumental phase
    1- Dispending a sample aliquot into a reaction vessel
    2- Combining the sample with one or more reagent
    3- Recording some physical/chemical consequence of the reaction
    4- Calculate the value of the quantity measured
    C. Post-instrumental phase
    1- Lab. staff accept the value (result) as being of good quality
    2- The report is sent to the requesting physician
    (III) Physician evaluates the report:
    A. Physician assesses whether the measurement could be consistent with other known patient information
    B. The physician makes a clinical decision at least partially based on the report measurement.


    INTRODUCTION TO QUALITY CONTROL

    Clinical laboratories perform quantitative, semi-quantitative, and quantitative tests on a variety of biologic specimens.
    The basic principles on quality controls were set by "Shewhart" in 1931, the ways in which these basic principles have been extended to develop systems of quality assurance have been revered by" Grannis" (1977).

    The immediate aim of quality control is assure that the end product of the analytical values regularly produced by a clinical laboratory are sufficiently reliable for their intended use, and to assure that the laboratory procedure analytical values that meet acceptable standard of precision, and accuracy at all times.

    To attainment of these aims requires that all lab. personnel, technologists, supervisors, and directors be knowledgeable of the causes of the analytical inaccuracies and of the techniques that available for their detection, correction, and control.
    Quality control in laboratory medicine has been defined as the study of those errors which are responsibility of the laboratory to recognize and minimize them.

    An alternative term "quality assurance" has been used to represent the techniques available to ensure with a specified degree of the confidence that the result reported by the laboratory is correct.
    In order to have such confidence, the laboratory director must be assured that there is both "precision control and accuracy control" performed.

    ♦ Quality control can be divided into two major types: Internal QC and External QC.





    Laboratory management

    The purely scientific and clinical approach to laboratory medicine is no longer sufficient. The bridge between the basic sciences and clinical medicine is now buttressed by essential support derived by form computer sciences , management techniques ,and industry. In recent years the relationship between laboratory medicine and industry has become bi-directional.
    In addition to the patient care service role , most individuals in laboratory medicine also have role as educator and many have roles as research and developmental scientists.
    To be successful person in laboratory medicine , must be skilled in all these functions and be aware of all those external influences affecting the practice of laboratory medicine.


    The five characteristics essential to success for an executive in medical laboratory :
    Motivation & skills.
    Vision & knowledge.
    Decision making ability .
    Good health & fitness (physically and mentally).
    Humility and recognition of other favor.

    The ten indicators of lack of management and communication skills in medical laboratory .
    Inability to maintain an adequate staff.
    Recurring or persistent misunderstanding with the hospital administration.
    Frequent or recurrent confusion concerning requisitions or reports of laboratory work.
    Frequent rush order for supplies.
    Low morale in the laboratory.
    Requests for deserved pay raise by competent workers.
    Excessive cost of operation.
    Ignorance of the cost of operation .
    Expenditure of much of the director's time in making decisions.
    Inability to do one or more tests when a "key individual" has a day off.




    Motivation:
    Maslon develop a theory of human motivation based of the belief that man is a wanting "animal".

    X- management theory :
    Based on the assumption that people dislike work ,that they have to be driven , threatened and punished to achieve goals, and that they lack ambition and want only security .

    Y- management theory:
    Based on assumption that work a natural need as natural as rest or play ; people don’t have to be threatened or forced to work , and people want responsibility in the proper environment when given the opportunity .
    Although theory X and theory Y may be considered as "extremes" in styles of management.

    Z- management theory:
    A new approach suggests that the involvement of workers is a key for increased productivity , motivation of employees may be enhanced by combination of trust , subtlety and intimacy provided by management.

    The director or supervisor must be able to use the appropriate management tool for the situation or person with which one is dealing at the moment.
    There are others management styles like, objective management and total quality assurance management.



    Personnel management:

    Policy manual:
    All laboratories should have readily available administration police manuals.

    Job description :
    Is a summary of all the important or significant fact about a particular job.
    Job description should have a clear and easily understood description for the duties and responsibilities for the employees, It also helpful to define the purpose of the job and to show it's relationship to other jobs in the organization.

    Position classification :
    Job description and class or kinds of the employees are the basis upon which the individual positions are assigned at the appropriate level (class).

    Orientation:
    The important step of introducing the employee to his/her new environment comes after the selection and hiring process. An orientation program is a probably on of the most overlooked tools available to the manager.

    In service continuing education:
    Because of the rapidly changing nature of laboratory medicine , it is essential to have a continuing education programs .
    The staff should be given the time and encouraged to attend appropriate meeting programs in laboratory medicine.

    Intra laboratory staff meetings:
    Meeting of the laboratory director ,supervisors , and staff should be held on a regular basis to discuss.



    (Administrative professional and technical problems:

    Periodic full laboratory staff meeting are also useful as a forum for discussing
    problems , new policies and procedures and planning ).

    Personnel records :
    Access to confidential and personal information should be limited to appropriate individuals.

    Evaluation:
    An important part of employee development , is evaluation of performance.

    Discipline and Dismissal :
    Because of the seriousness of some acts, the laboratory director or supervisors may elect the acceptable discipline actions depend on the type of infraction and the circumstances surrounding it , some acts needs to recommend dismissal of the employee immediately.
    However, the procedure for such dismissals would include consultation with personnel department .
    Success in handling disciplinary problems comes with consistency of approach , promptness and equity in dealing with all personnel.



    ANTICOAGULANTS

    Anticoagulants are substances (natural made e.g. heparin or industrial synthetic) used to prevent clot formation of the blood.
    Before taking a blood sample for haematologic analysis; it is important to mix the blood sample thoroughly. If the tube has been standing , this requires at least 60 gentle inversion of the tube or two minutes on a mechanical rotator.
    Precaution must be taken to prevent errors in analysis of blood cells as result of in vitro change in EDTA. blood.
    In blood kept at room temperature, swelling of erythrocytes between 6-24 hrs. raises the hematocrit and MCV. and lowers MCHC. and ESR.


    Ethylene diamine tetra acetic acid:
    - Acts as chelating agent by removal of calcium ions.
    - The most widely common used anticoagulant for haematologic procedures.
    - Useful for CBC, blood films, platelets count (because it prevents platelets clumping).
    - EDTA. Is not useful for factor V and VIII assay.



    * Preparation:

    □ LIQUID FORM:

    - Dissolve 3gm. of EDTA. salt in 100 ml. of 0.7% aqueous NaCl solution.
    - Use 0.5 ml. for each 9.5ml. of blood.


    □ DRAY SALTS:

    - Dissolve 10 gm. of EDTA. salts into 100ml. of distilled water.
    - Pipette 0.1 ml. of this solution into each container (vial) to be used for 5ml. of whole blood specimen collection.
    - Allow to dry at room temperature or into oven at low temperature , without cover, put cover after dry.



    2) HEPARIN:

    - Naturally producing by liver.
    - Neutralize thrombin which prevents the blood clotting mechanism.
    - Useful for red cell fragility test, electrolytes studies, L.E.cells.
    - Not useful in CBC, blood films.


    3) SODIUM CITRATE:

    - Sodium citrate combines with calcium to form insoluble calcium citrate.
    - 3.2% sodium citrate is the most commonly used for coagulation studies (1volume of anticoagulant to 9 volume of blood).
    - For ESR. ( 4 volume of tri sodium citrate to 1volume of blood).



    4) ACID CITRATE DEXTROZE (ACD):

    - Commonly used in blood banking.
    - 63 ml. of ACD. is sufficient for 450ml. of whole blood.


    5) OXALATE:

    - Sodium oxalate can be used in chemistry, haematocrite, coagulation tests.
    - Replaced by Sodium citrate.
    - Double oxalate is replaced by EDTA.



    PREPARATION OF BLOOD FILMS


    *THICK BLOOD FILM:

    - A large drop of blood is taken on the centre of a clean labeled slide ( a drop of finger puncture or from well mixed EDTA. tube of blood by capillary tube or pipette).
    - With an other slide corner spread the drop over 1/2 an inch square area (20 mm.) in diameter.
    - When dry the thickness should be such that printed litters can be seen through it.
    - Thick blood films used for detecting Malaria parasites and Microfilaria …..


    * THIN BLOOD FILM:

    Making of spreaders:
    - Select a slide with smooth edge ( a high quality polished side slides can be used).
    - Using a glass cutter, cut across a corner of the slide.
    - Break off the corner by holding the slide corner between piece of cloth.
    Making a thin film:
    - Put one small drop of blood on a clean (grease-free) labeled slide.
    - Hold the spreader at 30-45degree angle against the surface of the slide.
    - Move the spreader back to touch the drop of blood and allow the blood to extend along the edge of the spreader.
    - Push the spreader with a steady hand across the slide.
    - Air dry the film.




    FIXING OF THIN BLOOD FILMS:

    * With absolute methanol, by immersing in a container of absolute methanol for 2 minutes.
    * When absolute methanol is no available absolute ethanol can be used.
    * Methanol containing water must not be used to fix blood films.




    STAINING OF THIN BLOOD FILMS

    Romanowsky stain :-
    Stains contain Eosin Y (acidic) and Azure B and other thiazine dyes "Methyline Blue" .
    Eosin stains the basic components of blood cells e.g. haemoglobin stains Pink-Red and granules of eosinophils stains Orange -Red .
    Azure B and Methylene blue stain the acidic component of cells.
    Buffers:-
    The ideal PH for staining blood films is 6.8 and in order to maintains this , buffered distilled water is used .
    Buffer solution :-
    -sodium hydroxide (NaOH) 8gm.
    -distilled water 1000cc
    -Potassium dihydrogen phosphate (KH2Po4 ) 27.2gm.
    -distilled water 1000cc.

    Take 23.7 cc of solution 1 , add 50cc of solution 2.
    Take 20cc of distilled water.

    To make 1 liter PH 6.8 buffer water using Na2HPo4 and KH2Po4 :-

    Di-sodium Hydrogen Phosphate anhydrous (Na2HPo4)----- 0.47g
    Potassium di-hydrogen phosphate anhydrous (KH2Po4 )----- 0.46g
    Distilled water-----up to 1 liter.




    Giemsa stain 500 ml stock :-
    Giemsa powder---------------------------3.8gm
    Glycerol-----------------------------------250 ml
    Methanol----------------------------------250 ml
    weight giemsa powder and put it in 500 ml dry browen bottle with a few glass beads.
    measure the glycerol and add it to the stain powder ,mix it well .
    place the bottle of stain I water bath at 50-60 c for 2 hours.
    measure the methanol and add it to the stain mix well.
    label the bottle.
    filtrate the stain before using.

    Leishman stain 400 ml :-
    Leishman powder -------------------0.6g
    Methanol -----------------------------400ml
    weight the stain powder on a dark brown bottle with a few glass beads.
    measure the methanol and add it to the stain ,mix well.
    place the bottle at 37 c water bath to help to dissolve the dye.
    label it and keep it at room temp.
    filtrate the stain before using.



    New methylene blue (100 ml "1%w/v):-
    new methylene blue powder ----------------------1g.
    soudium citrate -------------------------------------0.6g
    sodium chloride ------------------------------------0.7g
    distilled water---------------------------------------100ml
    weight the sodium citrate an sodium chloride then dissolve both in distilled water.
    add the new methyline blue powder and mix it and put it in dark brown bottle.
    lable it and filtrate befor using.



    Wright's stain (400ml):-
    wright's stain powder -------------------------1g
    methanol --------------------------------------400ml
    weight the wright's stain powder and put in dark brown bottle with a few glass beads.
    measure the methanol and add it to the powder and mix it well.
    place the bottle in 37c water bath to help day to dissolve.
    label the bottle.
    allow 3-5 days before using the fresh stain , filtrate before using.





    Fields stain:-
    Stain A:
    methylen blue powder ------------------1.3g
    di-sodium hydrogen phosphate -------12,6g
    potassium di hydrogen phosphate ----6.25g
    distilled water ---------------------------500ml
    dissolve the methylene blue and di- sodium hydrogen phosphate in 50 ml of distilled water in flask.
    put the flask in water bath at 50-60c for half hour.
    dissolve the potassium dihydrogen phosphate in 450ml of distilled water and add it to the flask mix it well and keep it for 24 hour and filtrate.



    Stain B:
    eosin powder ---------------------------------1.3g
    disodium hydrogen phosphate ------------12.6g
    distilled water --------------------------------500ml
    dissolve the stain powder with the disodum hydrogen phosphate in 50 ml of distilled water.
    mix it well in water bath at 50-60c.
    add potassium di hydrogen phosphate to 450ml distilled water and add it to the flask.
    keep it for 24hour and filtrate befor using.
    For staining:
    Dip the dry thick film in methanol for 1-2 sec.
    Dip the smear in stain A for 1-2 sec then in stain B for 1-2 sec.
    Dip the smear at buffered water for a few second and allow to dry.


    Hb- estimation

    Hemoglobincyanide (HiCN) method :

    *Principle :-
    Blood is diluted in solution of potassium cyanide.
    The potassium ferricyanide oxidize hemoglobins to hemoglobin (Hi) "methonoglobin".
    The potassium cyanide provide cyanide ion (CN ˉ ) to form hemoglobincynide (HiCN) "cyanmethemoglobin" wich has abroad absorbation maximum at wave length 450 nm.
    The absorbation of the sokution is measured in photometre or spectrophptometer at 540nm and compared with that of a standerd HiCN solution.
    The diluents "Drabkin's solution"
    Potassium ferricyanide K3Fe(CN)6 ------------------0,200g
    Potassium cyanide KCN -------------------------------0,05g
    Dihydrogen potassium phosphate anhydrous
    KH2PO---0.140g
    Non- ionic detegrant ------------------------------------1.0ml
    Distilled water to ----------------------------------------1000ml
    *Substituting dihydrogen potaissum phosphate for sodium bicarbonate NaHco3 shotens the time needed for complete conversion of hb to HiCN from 10 min to 3 min.

    Method:-
    20ul of blood is add to 5 ml of drabkin's reagent .
    Mix well and allow at room temp. for at least 3 min .
    Read the absorbance at 540 nm against reagent blank.
    Measure the absorbance for HiCN standerd.
    Hb (g/dl)=(A sample/A standerd) *(standerd conc."mg/dl" / 100mg/g)*251
    A standerd curve can be prepared.




    Hematocrit (packed cell volume)

    Is the ratio of volume of erythrocyte to the whole blood.

    Procedure :-
    Mix the sample well .

    By using macro method (wintrobe's tube).

    Fill the tube to the 100mm. mark.
    Centrifuge at 2500 g for 30 min .
    Read and calculate as:
    HCT (PCV)=L1/L2.
    L1 =is the height of RBC column in mm.
    L2 = is the height of whole blood.

    By using micro method (capillary tube).

    Fill the capillary tube at least to 5 cm.
    Seal the empty end.
    Put the tube at microhematocrit centrifuge read with the sealed end away from the center.
    Centrifuge for 5 min at 10.000 to 12000.
    Read by a measuring device.


    Blood cells counting

    Any cell counting procedure is including three steps:-
    Dilution of the sample , sampling the dilution suspension into a measured volume and counting the cells in that volume.


    Counting can be with manually or semi-automated methods:-

    Electronic counting methods:

    Principle :-
    Electrical impedance:-
    Cell passing through an aperture through which a current is flowing; cause changes in electrical resistance which are counted as voltage pulses.

    Light scattering :-
    In electro optical counters a photomultiplier tube detects light scattering either external reflections from the surface of cells, from transmitted and refracted light passing through the cells, or from diffracted light which has passed tangential to cell surface.
    The output of photomultiplier tube is recorded by the pen on moving printing paper.




    Manual counting method:-

    WBC counting:
    Using improve neubaures counting chamber ( haemo-cyto meter).
    Sample should be EDTA anti-coagulated blood but not heparin or sodium citrate .
    Reagent:-
    Glacial acetic acid------------------------------------2ml
    1% aqueous sol. Gentian violet ---------------------1ml
    Distilled water ---------------------------------------100ml


    Procedure :

    Draw blood up to 0.5 mark of WBC pipette.
    Carefully wipe excess blood out said the pipette using tissue or gauze.
    Draw WBC reagent up to the 11 mark.
    Mix, after 5 min discard a few drops out said and fill the counting chamber by butting a drop on the edge of the cover slip.
    Read in WBC area the all four squares
    Calculate :
    WBC= (cell counted×20×106)/(4×0.1) or (No.counted cells)/2 = X
    Then X÷10 = WBC count × 109/L
    WBC = count × 50.

    Correcting of WBC counting if there is many nucleated RBCS.
    Corrected WBC = (WBC count ×100)/(Nucleated RBC+100)

    ☼ A blood film should stained to confirm the report.





    RBC counting using haemocytometer counting chamber:-

    RBC s diluting fluid :
    Sodium citrate ----------------------------3.0g
    Formalin ------------------------------------1.0ml
    Distilled Water ----------------------------100ml
    The solution is stable at room temp. for 1 year.

    Procedure:-
    By using RBC pipette draw blood up to 0.5 mark and carefully wipe the excess blood using cotton or gauze.
    Draw diluting fluid up to 101 mark .
    Mix for 5 minutes .
    Discard a few drops from the pipette .
    Introduce a small volume under the cover slip which is placed on the counting chamber .
    Allow the cell to settle down for 2-3 min .
    Examine it under the 40x objective microscope.
    Count the cells in the RBCs part and calculate as:
    RBC = (counted RBC×Dilution factor)/(Area counted×Depth of chamber)
    RBC=Count × 10,000

    In general:-
    Any cells counted /cu mm =count×10(depth factor)×area factor ×dilution factor.







    Platelets counting using counting chamber:-

    Reagent:
    1%amonium oxalate in D.W
    Keep the reagent in the refrigerator filtrate the needed amount before use and in the end of the day discard the used amount.
    EDTA sample is used.
    Perform Plts. Count within 2 hours of collection the venous blood sample.
    By using RBC pipette draw blood up to 0.5 mark and wipe the excess blood.
    Draw from the diluting fluid up to 101 mark and mix it well (blood is diluted 1:200).
    After 5 min. discard a few drops, then put a small drop on the counting chamber.
    Place the counting chamber in a Petri dish on a wet filter paper supported on tow sticks .
    To prevent drying of the fluid and cover the lid of Petri dish .
    Keep it for 15 min.
    Examine under 40x objective.
    Keep the condenser down and reduce the light.
    Count in Plts. In all 25 small squares.

    Calculating :
    Plt = 〖(count×20×10)/(0.2×0.1)〗^6
    Plt = (count×dilution)/(volume of fluid)
    Plt = No. × 1000




    RBC indices:

    MCV = mean red cell volume roviding information on red cell size .
    MCV(fl)= (HCT(L/L))/RBC×10

    MCH =Mean red cell haemoglobin :the amount of Hb in an average red cell.
    MCH(Pg) = HGB/RBC×10

    MCHC =Mean red cell haemoglobin concentration.
    MCHC(g/l) = HGB/HCT×10








    Count Terminology
    WBC >12×109/L Leukocytosis
    WBC <3.0×109/L Leukocytopenia
    Neutrophils >5.5×109/L Neutrophilia
    Neutrophils <2.0×109/L Neutrophilia
    Lymphocytes >75.5×109/L Lymphcytosis
    Lymphocytes <1.6×109/L Lymphcytopenia
    Monocytes >2.0×109/L Monocytosis
    Eosinophils >1.5×109/L Esinophilia
    Basophils >0.5×109/L Basiophilia
    Hgb <11.0g/dl Anemia
    RBC >6.0×1011/L Erythrocytosis
    PLTs <150×104/L Thrombocytopenia
    PLTs >500×104/L Thrombocytosis
    MCV >98 fl Macrocytosis
    MCV <7.7 fl Microcytosis
    RDW >16.5 % Anisocytosis
    WBCs <3.0×109/L
    Panocytopenia
    RBCs <3.0×1011/L
    PLTs <100×104/L
    MCH <27 pg Hypochromia[/LEFT]












    ESR:



    Erythrocyte Sedimentation Rate
    Non specific test, it is raised in wide range of infectious, inflammatory, degenerative and malignant conditions associated with changes in plasma protein.
    ESR also affected by many other factors including anemia, pregnancy, hemoglobinopathies, treatment with anti-inflammatory drugs.

    Principle:

    RBCs aggregate to form rouleaux formation and sediment through the plasma when citrated blood is positioned vertically.
    Fibrinogen, immunogloblins and c- reactive protein increase RBC aggregation .
    Methods:
    Westergren's method .
    Wintrobe's method.


    Westergren's method

    Westergren's pipette is about 30 cm long with a bore diameter of about 2.5 mm.
    The lower 20 cm are marked from 0-200.
    Anti-coagulant used is 3.8 % tri sodium citrate (1 part of anticoagulant to 4 parts of blood).
    Fill the pipette by sucking till 0 mark and clan pit vertical in the Westergren's rack .
    After 1 hour read the upper level of red cells.


    Wintrobe's method

    The Wintrobe's tube is about 11 cm long bore diameter is 2.5 mm and the bottom 10 cm are graduated.
    Graduation are from zero (top)to 100 (bottom).
    EDTA blood is used to fill the tube till zero mark .
    Set it up vertically and read after 1 hour.




    Stages of ESR:
    First 10 min: the period of aggregation and reuleaux formation occurs ,sedimentation is slow.
    Next 40 min. the period of fast settling "during this period fall rate is constant.
    Last 10 min. the final period of packing .
    Rapid ESR :

    In any chronic infection e.g. tuberculosis.
    Extensive inflammation , cell destruction or toxemia .
    Pregnancy ,after the second month .
    Active rheumatoid arthritis.
    Nephrosis.
    Active syphilis .
    Post operation states .
    Liver diseases .

    Slow ESR:

    New born infants.
    Polycythemia .
    Congestive heart failure .
    Sickle cell aneamia .
    Allergic states.





    Haematological study for CSF:-C.S.F appearance :-
    Normally the C.S.F is colourless .
    Purulent /cloudy :- Presence of pus cells.
    Bloody :- Due to a traumatic lumber puncture.
    Clot:- Indicates high protein concentration with increased fibrinogen as can occur with pyogenic meningitis or when there is spinal constriction .

    TOTAL AMOUNT OF C.S.F IN THE BODY :120-150 ml.
    Functions :-
    To protect central nervous system.
    Act as an exchange media between central nervous system and the blood.
    Glucose in C.S.F is roughly 50% lower than in the whole blood.
    Normal C.S.F glucose = 2.5 -4.0 mmol/L (45-72 mg/dl).
    Normal protein = 0.15-0.40 g/L (15-40 mg/dl).

    Cell count :
    Bloody samples or clot containing samples are unstable for cell count .
    (diluting fluid is isotonic 0.1% toluidin blue or isotonic methylene blue )
    Mix 1 drop of CSF with 1 drop of diluting fluid >
    Fill the counting chamber .
    Wait for 2 min.
    Examined microscopally .
    Calculate :
    (using improved neubures chamber cells counting in 4 large squares × 5)
    Report the number of cell / L=count×106 cells/L or cell/mm³.
    If using modified Fuchs-Rosenthal ruled chamber cells counted in 5 large squares multiply by 2.



    Reticulocyte count:-

    Reticulocytes are immature RBCs which still containing the remains of ribo-nuclear protein .

    1% brilliant crystal blue (supravital stain) .
    Brilliant cresylblue powder ----------------------------1g.
    Sodium chloride ----------------------------------------0.7g.
    Soudium citrate ----------------------------------------0.6g.
    Distilled water -----------------------------------------100ml

    Store in dark bottle under refrigeration .filter before use .
    Place 1 volume of stain in test tube .
    Place 1 volume of blood to the stain and mix .
    Incubate at 37c for 10-30 min .
    Mix and spread 1 drop of mixture to make a thin blood film.
    Examine with oil immersion lens ,count at least 500 cell and calculate the percentage.

    Retic.Count%= (Retic counted)/(Retic counted+RBC counted)×100

    The absolute number of reticulocytes can be calculated from the reticulocyte percentage and RBC count from an automated blood counter.
    Reticulocytes (×109/L) = (Reic.(%)×RBC(×〖10〗^12 /L))/100×100
    Normal values :
    Infant at birth: 2-6%
    Children up to 5 years : 0.2 -5%
    Adults : 0.2-2 %

    Reticulocytes index = (Retic.% ×HCT)/45



    Sickle cell tests
    :


    Sickling test (slide method)"Daland & Dasilva method":-

    A reducing agent is used "sodium meta-bisolphate" to deoxygenate the hemoglobin in red cells. Only deoxy sickle hemoglobin (Hb S) can produce the sickling shape change .
    Reagent :- (20% sodium metabisulphate):

    -Weight 0.2 g of sodium metabisolphate .
    -Add 10ml of distilled water or deionized water and mix until fully dissolved.
    -Use the reagent for only 24 hr.
    ☼ Daily preparation of the reagent.
    Producer :-
    -Place a drop of mixed blood on a slide and put a drop of the reagent and mix it well.
    -Put cover slip and seal the edges with wax or vaseline .
    -A negative and positive control should be prepared from a known samples or after Hb-electrophoresis .
    -Incubate the slides at room temperature for 4hr or at 37c incubator for 1-2 hr.
    -Examine the slides using 40x objective lens looking after sickle cells.
    -Report the result as negative or positive .

    ♦The test is not sufficiently sensitive to detect very small amount of Hb S which may present in the blood of infants (below 6 months age), when a patient has been transfused , the test should be performed after 2-3 months to obtain a reliable result.




    Hb S solubility test:-
    Dithionate tube test

    Red cells are lyses due to the phosphate buffer -saponin solution containing sodium dithionite , HbS is insoluble then the solution becomes turbid.

    Reagent :

    1-phosphate buffer – saponin :-
    Phosphate dihydrogen phosphate KH2PO4------33.78g
    di-potassium hydrogen phosphate K2HPo4------59.33g
    white saponin ---------------------------------------2.5 g
    distilled water -------------------------------------- 250 ml
    -Dissolve the salts in 150 mal of distilled water , carefully add the saponin , avoiding bubbles as much as possible you can .
    -Make up to 250 ml with D.W and mix gently , store at 2-8 c.
    -Measure 20 ml of buffer saponin solution .
    -Add 0.2 g of sodium dithionate and mix gently until dissolved .
    ▓ use the reagent only for 24 hr .

    Method:-
    -Add 20 μl of well mixed whole blood EDTA anticoagulated to 2ml of working reagent .
    -Allow a room temperature for 5 min.
    -Place the tube against a white paper or news print sheet .
    -Examine the turbidity .
    ◙ if there is turbidity ( news print not visible ) indicate the presence of HbS .
    ◙ clear solution indicates a negative test .
    ☼ All positive test should be followed by Hb-electrophoresis.








    Platelets (thrombocytes):
    Platelets have a short life (10 days).

    Function of platelets :
    1-Platelets adhesive to collagen of wound surfaces , probably Von Willebrawd Factor (vWF) act t bind platelets to appropriate sub-endotholial tissues .
    -The process of adhesion causes platelets to become activated and undergo the "release reaction " in which platelets discharge ADP , serotonin , epinephrine , Ca++ and fibrinogen .

    2-The product of the release reaction , particularly ADP , cause platelets to aggregate to each other and form the "haemostatic plug " ,strands of fibrinogen link adjacent platelets to each other and this linkage is strengthened by another plasma protein called thrombospondin .

    3-Following aggregation or stimulation by exposure to subendothelial tissues , platelets appear to shift coagulation F.V from their alpha granules to platelets surface . The activated F.V( F.Va ) acts as the binding site for F.Xa . This step localized haemostasis to the wound site and protects the growing clot from the action of inhibitors of coagulation .

    4- Coagulation produces thrombin which causes platelets to release the reminder of their stored material and undergo fusion , mating a firmer plug .

    5-The platelets extend dendritic process which intercept fibrin strands . Then the thrombasthenin of the platelets dendrites; which is similar to contracts, causing the clot to undergo clot reaction.




    Clotting factor :-


    Capillary fragility test of Hess:-

    (Kumpel –Leade sign , tourniquet test)
    1-inflate sphygmomanometer cuff around arm at 80 mm/Hg pressure for
    5 min .
    2- look for petechiae in an area 5 cm indiameter just below the elbow .
    3- inder normal circumstances the number of petechiae should be less than 5 . more than 5 indicate a positive test .

    A positive test may be found in reduced capillary resistance "or increased capillary fragility " as in non-thormocytopenic purpura and scurvy. It may also be positive in thrombocytopenia when the platelets count blow ≅70,000/cum.











    Bleeding time tests

    Duke's method:
    1- Clean the lobe of ear or tip of finger with alcohol and let it dray .
    2-for ear glass slid is placed behind the ear lobe and hold firmly in place, this provides a firm site for incision .
    3-Pierce the lobe for the ear by a firm stroke against the glass slide if the ear lobe has been increased , start the stop watch when the stab was made.
    4-Bleeding of the wound should be allowed to proceed without pressure and the blood is allowed to drop on the filter paper .the paper should be moved so that each drop will fall an afresh area , when blood slows , the wound is touched gently with a fresh area of the filter paper at 30 sec. intervals.
    When blood no longer stains the filter paper ,the watch is stopped and the time record.

    Normal values = up to 6 min.
    Over 10 min. is definitely abnormal.
    Precaution:

    -in children heel should be used .
    -in suspected cases of bleeding disorder the bleeding may not be controlled easily from the ear lobe lance fingertip puncture are better .
    -the area to be punctured should not be congested .
    -the size and depth of the wound may vary if one does not have a standardized technique .
    -if the bleeding persists for more than 15 min it should be stopped by placing a dray gauze and applying finger pressure .



    Ivy's method :-

    1-Clean the inner aspect of the forearm .
    2-Place a blood pressure cuff on the upper arm , inflate at 40 mm/Hg and maintain the same throughout the test.
    3-Select an area on the forearm velar aspect with is devoid of superficial veins . stretch the skin laterally between the thumb and forefinger and hold in a taut position.
    4-Using a lancet a skin puncture is made on ventral surface of the arm (3mm incision ).
    5-With filter paper the blood is gently blotted until bleeding ceases.

    Normal values : 2½ min. to 7 min.


    Clotting time test :

    Capillary tube method of Wright :
    Blood is collected in capillary tubes about 12 tubes from a finger prick made after aseptic precaution , the tubes are sealed with plasticine and immersed in water bath at 37c&ordm; . after 4 min. remove the first tube from the bath and expel the blood in it with one end immersed in dish containing water . Repeat this every 30 sec. with the other tubes till the blood is expelled in warm clot and note the time .

    * an alternative way determining the end point is to break the capillary tubes every 30 sec. until clot is seen between the two broken ends by these methods normal clotting time is 5-10 min. at 37c&ordm;.



    Lee and White's method

    1-Make a clean vein puncture with as little trauma to the connection tissue between skin and vein as possible.
    2-Timing is begun when the blood first enters the syringe.
    3-Drow 3-5 ml of blood and withdraw the syringe and the needle , disconnect the needle , place approximately 1 ml of blood in each of 3test tubes.
    4-Place the tubes in a stand and put them in water bath at 37c&ordm;. wait for 5 min. (if in room temp. wait for 10 min. )
    5-take the first tubes and gently tip it every 30 sec. to test for clotting , after every 30 sec. check another tube for clotting .
    6-Record the clotting time for each tube , sum all the times and divided by three .
    Normal at 37c&ordm; = 5-10 min.






    Prothrombin time
    When an excess of tissue factor and calcium are added to citrate plasma a clot is formed in approximately 12 sec. in normal plasma .
    This test not only to measure prothrombin , but it can be divided into three portions :
    Presence of factor 7 to interact with the added tissue thromboplastin.
    Conversion of prothrombin to thrombin .
    Conversion of fibrinogen to fibrin .
    On this basis, it is evident that other clotting factor are involved in addition to prothrombin.
    - Proaccelerin (factor V) , proconvertin (factor VII ) , fibrinogen (factor I) and Stuart-Prower factor (factor X ) all contribute to the clot formation .
    - Circulation anticoagulants as well as heparin also act in this system to block orderly clot formation .
    Sample : citrate plasma (1-9).
    Procedure :
    Pipette 200 μl of thromboplastin –calcium (PT reagent ) into test tube.
    Pipette 100 μl of pre warmed plasma into the PT reagent , then immediately start timing .
    The amount of clot formation can be detected by tilting motion and observing for clot formation , or by using a nichrome loop , inserted and with drown twice per second until clot forms.
    A fibrino-meter with automatic timer is a convenient device for clot formation measuring.

    Normal values :11-16 sec.

    INR=(Result of patient PT)/(Result of PT control)×ISI




    Partial thromboplastin time (PTT)
    Screening test for entire coagulation mechanism , excluding factor VII and platelets .
    Fresh citrated plasma provides all the factors necessary for all intrinsic pathway except calcium (removed by the citrate ) and platelets factor (removed by the centrifugation ).
    By adding calcium and cephalin (platelets factor " partial thromboplastin ") back to the plasma.
    Procedure :
    Mix PTT reagent ( platelin " Kaolin and cephalin mixture ")
    Pipette 100μl of the regent into test tube .
    Pipette 100μl of the plasma into the test tube .
    Incubate at 37&ordm; for 120 sec.
    After incubation add 100μl of CaCl2 0.025M .
    Start stop watch and observe the clot formation .
    Normal range : 25 -39 sec.




    Thrombin time ( T.T)- Measure of the time required for thrombin to convert fibrinogen to fibrin .
    - Can be useful to monitor heparin therapy and treatment of fibrinolytic agents e.g. streptokinase .
    - Some substances interfere with the action of thrombin on fibrinogen will prolong the thrombin time , such substances include the fibrinogen and fibrin degradation products produced in disseminated intravascular coagulation "DIC".

    Procedure:
    Add 200μl of plasma is incubate at 37&ordm; for 3 min. in a test tube/(cuvitte).
    Add 100μl of T.T reagent.
    Observe the time for clot formation .
    Normal range :
    16-22 sec. if 1:31 T.T reagent dilution .
    8-10 sec. if 1:11 T.T reagent dilution is used .





    Fibrinogen
    100μl of plasma is taken into 900μl of owrens buffer (1:9) dilution.
    200μlis taken from this dilution and incubate for 3 min. at 37&ordm;.
    Add 300μl of thrombin (Fibrinogen reagent ).
    Read the time need for clot formation .
    Read value from curve chart .

    Normal range :
    160 – 450 mg/dl .



    An automated analyzer for coagulation profiles.






    Safety in laboratory
    The purpose of safety policies is to provide a frame work for recognizing, evaluating and controlling hazards accident with laboratory operations. Implementation of safety plans depends on the cooperation of department chairpersons, lab staff and members of safety committees.
    The responsibility for safety must be shared by all, and we must work towards meting the safety standards with the common goals of promoting healthy safe environment for all.
    It is important that all lab personnel to understand the high risk
    of infection and injuries.
    There are many sources of risk in the clinical laboratory such as:
    * Infectious agents.
    * Wounds, cuts and needle breaks.
    * Fire and burns.
    * Electrical shocks.
    * Exposure to chemicals.
    * Suffocation.
    * Poisoning.
    * Exposure to radioactive materials.
    * Explosion.



    Administrative responsibilities:

    Each individual lab administration member is responsible for implementing all safety policies in his/her section.
    - The department head will assure compliance with existing health and safety policies by designating a Departmental safety coordinator.
    RESPONSIBILITES OF LAB DIRECTOR:
    - Implementing the safety policies.
    - Designating a Departmental safety coordinator.
    - Reviewing and granting approval for laboratory operation that involve particularly hazardous chemicals.
    RESPONSBILITES OF DEPARTMENTAL SAFETY COORDINATOR:
    - Assisting each lab member in implementing the safety policies.
    - Conducting inspections to ensure compliance with existing policies.
    - Reporting all accidents and potential safety problems.
    - Communicating information on health and safety to lab staff.





    RESPONSBLITIES OF DEPARTMENT CHIEF:
    - Implementing all health safety policies in the department.
    - Developing written safety procedures applicable to their workers.
    - Mandating laboratory practice and engineering controls that reduce the potential for exposure to hazards.
    - Informing all lab staff and students of the potential hazards associated with laboratory operations and procedures for dealing with accidents.
    - Assuring employee training.
    - Supervising that safe practice is employed.
    - Instructing the department staff on location and use of all safety equipment in the lab.
    - Reporting accidents and any other safety problems.






    RESPONSIBILITES OF EMPLOYEES:
    - Following all safety procedures specified in the laboratory safety manual.
    - Attending required safety training workshops.
    - Reporting accidents, unhealthy and unsafe conditions to the department supervisor or to the departmental safety coordinator.
    - Notify the department supervisor of any pre-existing health conditions that could lead to serious health situation in the lab.










    LABORATORY EQUIPMENTS FOR SAFETY REQUIRMENT
    Drench showers.
    Eye and face washer.
    First aid kits.

    Laboratory safety information.

    Fire alarm.

    Fire extinguisher and sand buckets.

    Fire blanket.

    Spill control materials.
    Door posting and other signs.


    Mechanical pupating aids.
    Sharps containers and "Glass Only Boxes".
    Floor drains and sinks traps.





    General laboratory safety procedures:-
    Know the materials you are working with (chemical, biological, radioactive …….).
    Know the location of safety equipments and emergency procedures in your area.
    Always wear appropriate clothing and personal protective equipments e.g. safety eye glasses, lab coats, gloves…….in the lab; Remove the personal protective equipments before leaving the laboratory.
    When working with hazardous chemicals use properly operating fume hood.
    No smoking, drinking and eating in the working area in the lab.
    Keep working areas clean.
    It is preferable to not work alone due to safety consideration.










    Personal protective equipments:-
    Eye and face protection:-
    Must be worn in lab when there is a potential for contact with hazardous chemical or other agents
    The type of protection needs depends on the hazard nature.
    Lab coat and other protective clothing:-
    Lab coats and shoes (not open sandals) should be worn when performing lab work.
    Additional personal protective equipments such as aprons may be necessary depending on the type of work.
    Gloves:-
    Gloves should be worn as a standard precaution in lab work.
    Gloves should be replaced immediately if they are torn.
    In situations involving extremely hazardous chemicals; a special high resistant protective gloves are recommended or double gloves can be used.
    Coats, aprons and gloves should be removed when leaving the lab.


    Laboratory ventilation:-
    In generally laboratory must be well ventilated.
    All work with corrosive, flammable, odoriferous, toxic or other dangerous materials should be conducted only in a properly operating fume hood, gas cabinet or glove box.
    In special situations; vacuum systems are acceptable.









    Emergencies and accidents:-
    In order to be prepared for an emergency; know the hazards of each operation and compounds you work with.
    Asses the risk before using any tools or/and chemicals; and consider the following:-
    Sharp tools, needles, glass ware ….
    Moving mechanical arms and props.
    Heavy and/or unstable placed objects.
    Toxicity, reactivity, and flammability of the compounds.
    Amount of chemicals involved.
    The expected exposure to the chemical compounds.
    Potential routes of entry to body for the chemicals e.g. skin, inhalation, ingestion, contact….
    Source of heat.
    Electrical connection and cables.
    Gas cylinders.




    Laboratory safety training:-
    Laboratory director is responsible for insuring that lab employees receive proper training in safety plans and fire safety procedures.
    When bio-hazardous or radioactive materials are being used; additional training is required.
    Laboratory safety training will include:-
    Details, location and availability of laboratory safety plan.
    Basic toxicology, including rotes of entry and occupational exposure limits.
    General information on hazardous of chemicals.
    Proper use of fume hoods.
    Hazardous waste management.
    Fire safety training.
    The laboratory director and the safety coordinator are responsible to write safety policies, training programs, reporting and inspection.
    The safety manual must contain and explain:-
    Precaution signs and symbols.
    Safety equipments.
    Housekeeping plan.
    Bio-hazardous waste management.
    Personal protective equipments.
    Chemical safety policy.
    Emergency procedures.



    Working alone policy

    For safety reasons; working alone should be avoided.
    If nature of your work makes it un avoidable, take measures to ensure that others are aware of your location and have someone chick in with you from time to time, either in person or by telephone.
    Before conducting any work alone; your supervisor should be aware of your presence in lab.
    Assess the hazardous before start working.
    Your lab door should have a viewing window.
    At emergencies; you should have access to telephone, first aid kit, fire extinguisher and spill kit.




    Minor spill:-
    If the spill is minor and known limited danger, clean up immediately.
    During cleaning, wear appropriate protective apparel.
    The protective clothing required will depend upon the materials spilled, amount and airborne concentration.
    Cover liquid spill with compatible absorbent material such as spill pillow….
    A spill kit should be use.
    Soak towel in proper disinfectant such as a fresh 1:10 bleach solution can be place over the spill area for 10-20 min.
    Powdered materials should be covered with wet proper towels (if compatible) to avoid dispersal.
    Corrosive materials should be neutralized prior to absorption.
    Clean spills from the outer areas first, clean toward the center.
    Place the spilled materials into an impervious container, seal and dispose it properly.
    If appropriate, wash the affected surface with suitable detergent, mop up the residues.
    Appropriate solvent e.g. xylene; maybe necessary to clean surfaces contaminated with a non-water soluble materials.





    IF WORKING WITH HUMAN BLOOD:-
    The below precautions should be practiced:
    Use appropriate barrier precautions to prevent skin and mucous membrane exposure when contact with blood is anticipated, always wear gloves, mask and protective eye glasses or face shields to prevent exposure to the eyes, mouth and nose during procedures that are likely to result in droplets of blood; and wear gowns or aprons during procedures that are likely to result in splashes of blood, REMOVE ALL PROTECTIVE CLOTHING BEFORE LEAVING THE LABORATORY.
    Wash hands and other skin surfaces immediately if contaminated with blood and after the removal of gloves.
    Limit the use of needles to where there is no alternative and take protections to prevent injuries: needle should not be recapped, bent, removed from the syringe, or manipulated by hand; place needles and other sharps into puncture-resistant containers.
    Keep all specimens of blood in well contrasted containers with a secure lid to prevent leakage during transportation.
    Use biological safety cabinets whenever procedures that have a high potential for generating droplets are conducted.
    Never mouth pipette.
    Decontaminate lab work surfaces after spill of blood and when work activities are completed.



    Biological Hazards control
    Personnel who work in biological laboratories may handle infectious agents in addition to other hazards such as chemicals and radioactive materials.
    Over the years, there have been many ********ed cases of lab personnel acquiring diseases due to their work with infectious agents. Only approximately 20% of these cases have been attributed to a specific incident, with the rest assumed to be related to work practices in the lab, primarily the creation of aerosols.
    Whenever work with infectious agents is performed, all appropriate steps must be taken to protect personnel and the environment.
    There are basically four routes of exposure or four ways in which a person can come in contact with infectious agents. These routes are contact with the skin or mucus membranes, ingestion, inhalation, and inoculation. Each of these routes of exposure is discussed below.
    Contact with skin or mucus membranes:
    Spilled material can come into direct contact with the skin as can droplets produced by pipetting, removal of screw caps, and vortex mixing of unsealed tubes.
    The control of a contact exposure is accomplished through: 1. wearing of appropriate protective clothing such as a face shield, gloves, safety glasses, masks, and laboratory coats. Other ways to control contact exposure include using absorbent paper on the work bench, performing all procedures carefully, and frequently wiping work surfaces with a disinfectant.
    2. Keep all non-essential items away from the area where work is being performed to protect personal items from contamination.
    All contaminated wastes must be handled and stored properly to prevent contact exposure of lab personnel as well as housekeeping staff and waste handlers.




    Ingestion:
    Ingestion may occur either directly or indirectly. Exposure may occur from mouth pipetting or splashing from a container into the mouth or by contaminating the hands and then touching the mouth or items such as a coffee cup, food, or lip balm, that go into the mouth.
    The control of an ingestion exposure is accomplished through the use of mechanical pipetting devices whenever pipetting and by practicing good personal hygiene, such as washing hands frequently throughout the day and not eating or drinking in the work area. Food items also cannot be stored in refrigerators that contain hazardous materials or in the lab where work with infectious agents is being performed.

    Inhalation :
    It is generally known that aerosols are the primary means by which infectious diseases are spread and contracted. An aerosol can be either a liquid or a dry particle. An aerosol with a diameter of five microns or less can easily be inhaled and carried to the alveoli of the lungs. These aerosols can remain airborne for a long period of time and can spread wide distances, especially after entering the ventilation system. Particles with a diameter larger than five microns tend to settle rapidly and can contaminate the skin or other surfaces.
    There are many commonly performed procedures in the lab that can create aerosols. Examples include centrifuging, heating inoculating loops, using a blender, blowing out the last drop in a pipette, and changing animal bedding.
    The control of inhalation exposure is accomplished by a combination of using the appropriate safety equipment such as biological safety cabinets and by performing procedures carefully to minimize the creation of aerosols.









    Inoculation:
    Inoculation in a lab usually occurs with a needle and syringe. Exercise extreme caution whenever using a needle.
    Restrict needle use; whenever an alternative to a needle is possible, it should be used.
    Inoculation can also occur through animal bites and other sharps such as Pasteur pipettes and razor blades.
    The control of an inoculation hazard is accomplished by the safe use, handling, and storage of needles and other sharps.
    After using a needle, do not recap, bend, break, remove it from the syringe, or manipulate it in any way. Many people have been accidentally stuck with a needle during the process of recapping it.
    The needle and other sharps should simply be placed into a sharps container to prevent any injuries.













    Housekeeping

    a) Work areas must be kept clean and free of obstructions.

    b) Stairways and halls must not be used for storage. This applies to both equipment and personal property.

    c) Walkways and aisles in laboratories must be kept clear.

    d) Access to emergency equipment or exits must never be blocked.

    e) Equipment and chemicals must be stored properly.

    f) Spilled chemicals must be dealt with immediately and if safe cleaned up by the chemical user. Spills must be reported to the supervisor.

    g) Wastes must be placed in appropriate, labeled containers.

    h) Materials no longer used must not be allowed to accumulate and must be disposed properly.

    i ) Floors should be cleaned regularly (24X7) using a suitable detergent.




    Hazardous Waste Disposal

    Waste minimization
    In order to minimize the amount of hazardous waste presented for disposal, it is important to follow these guidelines:
    Avoid overstocking: one of the main sources of laboratory waste is surplus stock - the result of over buying.
    Do not accept donations of materials that you don't plan to use. Many companies have traditionally unloaded unwanted reagents by donating them to laboratories, which eventually transfers the cost of disposal to the laboratory.
    Substitute non-hazardous experimental materials for hazardous ones.
    Hazardous waste disposal guidelines
    Label all waste materials completely and legibly, using labels available from the Waste Management Program. Inadequately labeled containers will not be accepted.
    Package waste materials in approved containers.
    Over filled and/or leaking containers cannot be accepted for disposal.
    Never discharge wastes into the sewer unless you have verified that hazardous wastes regulations permit you to do so.



    Chemical waste


    Organic solvents and oils
    Collect in the containers provided by the Waste Management Program.
    Indicate the composition of the contents as accurately as possible on the attached label.
    Unknown chemicals cannot be accepted.
    Peroxide-forming and explosive chemicals
    Do not mix with solvents or other waste.
    If the material is older than one year, do not attempt to open or move the container.
    Corrosive and acids
    Collect acids (pH<7) and bases (pH>7) separately in the plastic containers provided by waste management program. Do not mix acids with bases.
    Indicate the composition of the contents, as accurately as possible, on the attached label.




    Material Safety Data Sheets

    In general, a MSDS consists of several information:

    1. Chemical Identification 2. Composition/ Information on Ingredients 3. Hazards Identification
    4. First-Aid Measures 5. Fire-Fighting Measures 6. Accidental Release Measures 7. Handling and Storage 8. Exposure Controls/Personal Protection
    9. Physical/Chemical Properties 10.Stability and Reactivity 11.Toxicological Information 12.Ecological Information 13.Disposal Considerations 14.Transport Information 15.Regulatory Information 16. Other useful Information


    Biomedical waste

    1- Animal carcasses
    Place in the plastic-lined biomedical waste containers provided by the Waste Management Program.
    Store in a refrigerated area.
    2- Infectious laboratory waste
    Place in the plastic-lined biomedical containers provided by Waste management program.
    3- Blood and blood contaminated materials
    Un- clotted blood can be disposed of via the sanitary drains. Designate one sink for this purpose.
    After discharging blood, decontaminate the sink with a 5-10% dilution of household bleach. Allow a contact time of 20 minutes; then rinse with water.
    Dispose of blood-contaminated materials as infectious laboratory waste.






    Bio-medical sharp waste
    Sharps are defined as any material that can penetrate plastic bags: examples include syringe needles, scalpel blades, glass and plastic pipettes, disposable pipette tips, etc.
    Contaminated sharps
    Label a plastic, puncture proof container (e.g. empty liquid bleach bottle) with the word "SHARPS", the appropriate hazard warning symbol (e.g. biohazard, radioactive).
    Discard containers of sharps contaminated with infectious materials into biomedical waste containers as per the procedure for infectious materials waste.
    Discard containers of sharps contaminated with radioactive materials as per the procedure for solid radioactive waste.
    Non-contaminated sharps
    Label a puncture-proof container (wide-mouth plastic bottle or a heavy-duty cardboard box lined with plastic) with the word "SHARPS".
    Accumulate in the designated container, without overfilling.
    When full, close and seal the container and place it beside the regular garbage receptacle for pickup by the cleaning staff.




    Broken glassware
    It is more preferable to deal with it as in the sharps.
    Empty chemical reagent bottles
    Remove the cap from the empty bottle and allow volatile materials to evaporate into the fume hood.
    Rinse the bottle three times with tap water and let dry.
    Remove or obliterate the label.
    Place the uncapped bottle next to the garbage receptacle.
    Radioactive waste
    Solid waste (except sealed sources)
    Whenever possible, package alpha emitting radioisotopes separately from other radioisotopes.
    Whenever possible, package long-lived (half life > 10 years) radioisotopes separately from short-lived radioisotopes.
    Accumulate wastes in the solid radioactive waste containers provided.
    Update the information on the label as wastes are placed in the container.
    Sealed and encapsulated sources
    Do not package sealed sources with other types of waste materials.
    Contact your local Hazardous Waste Coordinator.


    Liquid scintillation vials
    Leave fluids in their vials.
    Deposit vials into the designated drum in your building's waste storage area and enter the required information on the inventory sheet attached to the drum.
    Liquid radioactive waste
    Aqueous liquid wastes at or below 0.01 scheduled quantity per liter can be disposed of via the regular drain
    N.B. It is illegal to dilute for the purposes of reducing radioactivity to below this level. Consult with the Radiation Safety Officer if additional assistance is required in determining scheduled quantities.
    Containers are available for laboratories that are unable to avoid the generation of liquid radioactive wastes. In order to control costs, you are asked to exercise great care to fill the containers with only such materials.








    Fire safety
    Laboratory fires can by caused by bunsen burners, runaway chemical reactions, electrical heating units, failure of unattended or defective equipment, or overloaded electrical circuits. Familiarize yourself with the operation of the fire extinguishers and the location of pull stations, emergency exits and evacuation routes where you work. In the event that the general alarm is sounded use the evacuation routes established for your area and follow the instructions of the Evacuation Monitors. Once outside of the building, move away from the doors to enable others to exit.

    1- The fire triangle
    Fire cannot occur without an ignition source, fuel and an oxidizing atmosphere (usually air), the three elements that comprise what is called the "fire triangle":

    Fire will not be initiated if any one of these elements is absent, and will not be sustained if one of these elements is removed. This concept is useful in understanding prevention and control of fires.


    2- Classes of fire
    The National Fire Protection Association (NFPA) has defined four classes of fire, according to the type of fuel involved. These are:
    Class A : fires involve combustibles such as paper, wood, cloth, rubber and many plastics.
    Class B: fires entail burning of liquid fuels like oil-based paints, greases, solvents, oil and gasoline.
    Class C: fires are of electrical origin (fuse boxes, electric motors, wiring).
    Class D: fires encompass combustible metals such as magnesium, sodium, potassium and phosphorus.
    3- Fire extinguishers
    Fire extinguishers are rated as A, B, C or D (or combinations of A, B, C and D) for use against the different classes of fires. Familiarize yourself with the fire class ratings of the extinguishers in your work area so that you will know what types of fire you can attempt to extinguish with them.
    Learn how to use the extinguisher in your lab, as there will be no time to read instructions during an emergency. Attempt to fight small fires only, and only if there is an escape route behind you. Remember to have the extinguisher recharged after every use: If you do fight a fire, remember the acronym "PASS" when using the extinguisher:
    P: Pull and twist the locking pin to break the seal.
    A: Aim low, and point the nozzle at the base of the fire.
    S: Squeeze the handle to release the extinguishing agent.
    S: Sweep from side to side until the fire is out.
    Be prepared to repeat the process if the fire breaks out again.






    Preventing fires
    Use the following precautions when working with or using flammable chemicals in a laboratory; keep in mind that these precautions also apply to flammable chemical waste.
    Minimize the quantities of flammable liquids kept in the laboratory.
    Except for the quantities needed for the work at hand, keep all flammable liquids in approved flammable liquid storage cabinets. Keep cabinet doors closed and latched at all times. Do not store other materials in these cabinets.
    Use and store flammable liquids and gases only in well-ventilated areas. Use a fume hood when working with products that release flammable vapors.
    Keep flammable solvent containers, including those for collecting waste, well capped. Place open reservoirs or collection vessels for organic procedures inside vented chambers.
    Store flammable chemicals that require refrigeration in "explosion-safe" (non-sparking) laboratory refrigerators.
    Keep flammable chemicals away from ignition sources, such as heat, sparks, flames and direct sunlight. Avoid welding or soldering in the vicinity of flammables.
    Bond and ground large metal containers of flammable liquids in storage. To avoid the build-up of static charges, bond containers to each other when dispensing.
    Use portable safety cans for storing, dispensing and transporting flammable liquids.
    Clean spills of flammable liquids promptly.

    Evacuations
    In the event that the general alarm is sounded, follow the evacuation routes established for your area; do not use the elevators. Follow the instructions of the Evacuation Monitors. Once outside the building, move away from the doors to allow others to exit.[/CENTER]
    التعديل الأخير تم بواسطة soma; الساعة 03-10-2008, 01:28 PM.

  • #2
    الموضوع مفيد وشامل ........شكرا لك
    http://<a href="http://www.baby-gaga...AN.png</a></a>

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    • #3
      يعجز اللسان عن تعبيره بشكرك
      http://upload.wikimedia.org/wikipedi.../76/Mendel.png
      يمضى الزمان ويذكر التاريخ أفضل الاعمال

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      • #4
        موضوعك جميل جدا يامعلمي

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        • #5
          great job
          thank u so much

          god bless u
          happy eid
          ان عشت فعش حرا أومت كالاشجار وقوفا


          sigpic

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          • #6
            بسم الله الرحمن الرحيم

            ماشاءالله عليك اخي موضووووووع متكامل والكامل الله سبحانه وتعالى

            كل الشكر لك بعد الله سبحانه وتعالى
            - من جن بالحب فهو عاقل ومن جن بغيره فهو مجنون

            -إذا أحبك مليون فأنا معهم.. وإذا أحبك واحد فهو أنا ..وإذا لم يحبك أحد فاعلم أنني مت.:sm184:.

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            • #7
              المشاركة الأصلية بواسطة otainy مشاهدة المشاركة
              موضوعك جميل جدا يامعلمي
              شكرا لك يا أستاذ محمد

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              • #8
                اللهم لا علم لنا الا ماعلمتنا


                مشكور أستاذي العزيز فهد قحل

                كفيت ووفيت

                مشاركتك سوف تغني كثير من الطلاب عن تعب البحث في كتب علم الدم المشهورة وبعضها المعقد.

                أرجوا من الله العلي العظيم أن يوفقك ويجعل مجهودك هذا ذو فايده للطلاب والباحثين ويجعله في ميزان حسناتك

                :thumbs_up:

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                • #9
                  جزاك الله الجنة اخي فهد السعودي
                  فهد العراقي

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                  • #10
                    استاذ فهد
                    الاخوة الاعضاء
                    اناطالبب ماجستير امراض دم موضوعي
                    AMI hematotological and hemostatic changes in Acute myo. inf
                    ارجو مساعدتي اذا امكن
                    وجزاكم الله خير

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                    • #11
                      الموضوع عموما القريتو فيه وسط ومعلومة يا اخ الفلم فى الهيماتولوجى مابيصبغ بالفكس لان لشمن استين فيها من مكوناتها فكساتف و الجمسا استين بتحتاج فكساتف وهى سيئة لانها بتدى بك قراوند عموما الاخت سومة هى المشرفة مفروض تقدم النصيحة
                      اللهم انفعنا بما علمتنا
                      ومما ذادنى شرفا وتيها وكدت باخمصى اطاء الثريا دخولى تحت قولك يا عبادى وان صيرت احمد لى نبيا

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                      • #12
                        عزيزي فهد حقيقية تعجز الكلمات ان تعبر عن ماقدمته
                        واختصرها واقول لا تنسوا اخوكم فهد من الدعاء
                        اسأل الله العلي القدير ان يجعل عملك هذا في ميزان حسناتك

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                        • #13
                          مافي موضوع بالعربي

                          وشكرا

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                          • #14
                            جزاك الله خير الموضوع رائع

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                            • #15
                              THANX ALOT

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