Introduction to Practical Haemmatology 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 COMPONENTMainly 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 MANAGEMENTThe 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 olderinfants 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 measurin g 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 thepuncture 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 compou nds,….- 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.□ FLUOROME RTRY: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 throughthe 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 phase1- Preparation of the patient.2- Obtain the specimen3- Processing the specimen4- Storing the specimen prior the measuring steps.B. Instrumental phase1- Dispending a sample aliquot into a reaction vessel2- Combining the sample with one or more reagent3- Recording some physical/chemical consequence of the reaction4- Calculate the value of the quantity measuredC. Post-instrumental phase1- Lab. staff accept the value (result) as being of good quality2- 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 informationB. The physician makes a clinical decision at least partially based on the report measurement.INTRODUCTION TO QUALITY CONTROLClinical 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 managementThe 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 discussingproblems , 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.ANTICOAGULANTSAnticoagulants 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 Microfila ria …..* 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 FILMSRomanowsky 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)----- 0.47gPotassium di-hydrogen phosphate anhydrous (KH2Po4 )----- 0.46gDistilled water-----up to 1 liter.Giemsa stain 500 ml stock :-Giemsa powder---------------------------3.8gmGlycerol-----------------------------------250 mlMethanol----------------------------------250 mlweight giemsa powder and put it in 500 ml dry browen bottle with a few glass beads.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 -------------------0.6gMethanol -----------------------------400mlweight 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 -------------------------------------0.6gsodium chloride ------------------------------------0.7gdistilled water---------------------------------------100mlweight 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 -------------------------1gmethanol --------------------------------------400mlweight the wright's stain powder and put in dark brown bottle with a few glass beads.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 ------------------1.3gdi-sodium hydrogen phosphate -------12,6gpotassium di hydrogen phosphate ----6.25gdistilled water ---------------------------500mldissolve 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 ---------------------------------1.3gdisodium hydrogen phosphate ------------12.6gdistilled water --------------------------------500mldissolve 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.。