This action might not be possible to undo. Are you sure you want to continue?
Correct patient identification 2. Correct specimen identification 3. Gloves must be worn all the times 4. Clean the puncture site thoroughly 5. Dispose all sharp objects in puncture-resistant containers Factors to be considered before blood collection: 1. Type of test 2. Age of patient 3. Condition of patient 4. Skill of phlebotomist General Methods for Obtaining Blood 1. Skin puncture/ Capillary puncture 2. Venipuncture 3. Arterial puncture Skin Puncture Mixture of arterial and venous blood Increased pressure in arterioles yield a specimen rich in arterial blood Also contains interstitial and intracellular fluids Application of Microsample Collection Infants less than 6 months In young children In adult with poor veins o Extreme obesity o Severe burns o Geriatric patients o Patients with thrombotic tendencies Sites of Puncture Palmar surface of the 3rd or 4th finger Margins of the earlobe Plantar surface of the heel (fleshy part) or big toe Advantages of skin puncture using the finger Easily accessible to the medtech Less intimidation Easy to manipulate Ideal for peripheral blood smears Advantages of skin puncture using the earlobe Less painful due to lesser nerve endings Lesser tissue juice contamination More free flow of blood due to thinner skin
Ideal when searching for abnormal cells (ex. Histiocytes in bacterial endocarditis)
Disadvantages of skin puncture Less amount of blood can be obtained Blood obtained by skin puncture hemolyses easily Additional and repeated test cannot be done Sites to Avoid in Skin Puncture Cold and cyanotic areas (less blood flow) Inflamed and pallor areas (increased WBC due to chemotaxis) Congested and edematous area (increased tissue fluids) Scarred and calloused areas (cannot reach the capillary bed) Things to remember in doing the skin puncture Recommended depth of puncture o for children and adults : 2.4-3.0 mm o for infants: 1.6 mm Avoid excessive massaging Thumb, earlobe, and big toe should not be used as sites of puncture in adults Fingers must be dry before the test is performed Collect platelet count and blood smear first Venipuncture Three Factors Involve in a Good Venipuncture The phlebotomist The patient and his veins The equipment needed Methods of Collection Syringe Method Vacutainer/ Evacuated Tubes Butterfly infusion set Advantages of Vacutainer Method over syringe method: Safer method of blood collection as samples are taken directly into labeled tubes Offers a wide range of tube size and contains anticoagulant Avoidance of glass syringe breakage Commonly Used Anticoagulants EDTA o generally available as disodium, dipotassium or tripotassium salt of ethylenediaminetetraacetic acid o prevents coagulation by binding to calcium
3. increased MCHC. Large amounts of blood can be obtained for a variety of tests. more homogenous nuclei. Blood collected is ideal for blood chemistry and other serological test. Fainting Local Delayed Complication 1. Requires more equipment. In patient’s arm receiving an intravenous infusion Complications in Venipuncture Local Immediate Complication 1. which may be due to: Nervousness excessive pull of the plunger hitting past the wall of the vein in case of sclerotic and movable vein or hitting the vein through and through 3.discoloration and inflammation of surrounding tissues due to extravasation of blood 2. 4. Scars 4. Areas to be Avoided in Venipuncture 1. less painful. Thrombophlebitis. 18 months up to 3 years old o Femoral vein o Long saphenous vein o Popliteal vein o Ankle vein In 3 years old to adult life o Wrist vein o Dorsal veins of the hand o Veins on the antecubital fosa Median basilica vein Median cephalic vein . Thrombosis of the vein. Circulatory 4. increase OFT. Hematomas 2. Additional and repeated test can be done. helps platelets retain their functional capabilities o Buffered sodium citrate (0. 2.109 M (3. prominent. Hemoconcentration 2. breaking up of platelets (falsely high platelet count) o After 3 hours of EDTA at room temp WBC vacuolation of cytoplasm. not movable. Blood can be transported and stored for future use. irregularly or poorly defined cytoplasmic borders. Disadvantages of Venipuncture 1. Edema 5. decrease ESR Sodium Citrate o Used for coagulation studies in concentration of 1 part 0. bigger lumen. children and obese individuals. Side which mastectomy was performed 6. 3. 5. More complications may rise 4. Serum hepatitis Sites of Venipuncture: In newborn infants up to 18 months old o External jugular vein o Temporal vein (scalp vein) o Superior longitudinal sinus In older children. Failure of blood to enter the syringe due to collapsed vein. Fastest method of collecting blood samples from a large number of patient. Hematomoa. Difficult to do on infants. Burns 3. prevents formation of artifacts and can be used in preparing blood smears 2-3 hours after collection o excess EDTA results to: shrinkage of RBC’s = decrease spun Hct. falsely low ESR (no effect on Hgb) degenerative changes in WBC’s :. palpable Advantages of Venipuncture 1. 2.109 M + citric acid) may increase the stability of factors V and VIII Heparin o May be used in concentrations of 15-30 units/ml of whole blood o Interacts with antithrombin III and subsequently inhibits thrombin o Causes clumping of WBC’s and platelets o Causes blue coloration in a blood smear after staining with Wrights Stain o o Advantages: stable.inflammation of vein due to trauma wherein thrombus is present General Delayed Complication 1.2%) sodium citrate to 9 parts of whole blood o Binds calcium in blood. Requires more time and skill on the part of the phlebotomist.formation of small blood clots at the site of puncture 3. dev’t of irregularly shaped nuclei Platelets slowly swell and disintegrate o After 6 hours of EDTA at room temp RBC swell= increase MCV.
coagulation tests (PT. WBC. Serum to be tested for agglutinins should be separated as soon as possible and should not be stored in the refrigerator in contact with RBC. aPTT. 3. eosinophil count. Hemoglobin 4. Hayem’s fluid 2. 1. RBC. Cheap and economical 4. Drug and Radiation Therapy monitoring 3. It should by isotonic for RBC and hypotonic for WBC 2. 2-3% Glacial Acetic Acid 2. Gower’s fluid 3.disposable diluting pipette which consist of a uniform bore glass capillary pipette capable of measuring accurately and precisely various volumes of blood 2. Stored anticoagulated blood must be thoroughly mixed on blood rotators at least 2 minutes (or 60 inversions) after it has reached the room temperature. each having a specified number of blood cells 2. with high specific gravity 8. Blood specimen for platelet count. EXERCISE 3-6: Blood Diluting Fluids. CSF and other body fluids Diluting Fluids .8% Sodium Citrate WBC Diluting Fluids 1. Bethel’s 5. Qualitative Platelet Count Importance of CBC 1. If possible. Indicator of patient’s progress in certain disease states Hemocytometry . Hematocrit 5. 1. For plasma Hemoglobin. Turbidimetric Method o an unknown sample is compared to a set up of different solutions of turbidity. it should be done immediately. With buffer action 7. WBC differential Count 6. RBC Count 2. 3.5. 11 WBC count. RBC and WBC Count Complete Blood Count 1. AIDS Storage of Specimens 1.0. direct platelet count. Screening Procedure 2. Easy to secure and prepare 3. Specimens that cannot be examined immediately should be stopped and placed in the refrigerator (prevent decreased in pH) Ex. etc. With preservative action 6. Stable 5. Turk’s Solution . Hemocytometry. Unopette. 1% HCl 3. Manual/Microscopic o for cell counts of body fluids.plasma should be separated from other blood components before it is refrigerated 2.5. Do not shake 4. manual pipette (RBC and WBC pipette) Comparison of RBC ad WBC Pipette RBC Size of bulb Color of bead Volume in bulb Size of bore Calibration Principal use Larger Red 100 Smaller 0.0.used to dispersed blood cells to facilitate counting Properties of an Ideal Diluting Fluid 1. Formol Citrate (Dacie’s Fluid) 6.the estimation of the number of blood cells in a known volume of blood Methods: 1. and platelets o counts expressed on cubic millimeter due to the linear dimensions of the counting chamber Pipet 1. WBC count (leukemia) WBC Smaller White 10 Larger 0. ESR. 101 RBC count.2. Normal Saline Solution 7. WBC Count 3. non-allergenic and non corrosive RBC Diluting Fluids 1.) should not be stored longer than 2 hours. Toisson’s 4.
Technicon Autoanalyzer 2.repeat counts and use of the second value Increase WBC count. packed cell volume. and MCH Errors in Automated Counting Low RBC and WBC count. Presence of clots in the dilutions 6. Faulty filling of counting chamber o overfilling or underfilling of counting chambers o pressure of trapped bubble 5. Incorrect computation and reporting of final counts 11. MCV calculates PCV or Hct.due to turbidity of the solution Increased Hgb. Chipped or broken pipette 14. Fisher autocytometer. Defective microscope . Uneven surface of coverslip and counting chamber 13.cell passing through an aperture causes changes in electrical resistance which are counted as voltage pulses and will be recorded as voltage impulses in a photomultiplier tube and appears on . Sysnex o Electronic Particle Counter determines directly the concentration of: WBC. MCV. ratio of RBC volume to that of the whole blood o o Multiply by 106 if reported in SI units Allowable difference in cell counts per chamber is 15-16 cells o o o Multiply by 106 if reported in SI units Even distribution of cells in all four large squared (not more than 10-12 variation between squares) 10% agreement in repeat counts Sources of Errors in Blood Counting 1. MCHC. Uneven distribution of cells 9. Results are interpreted in a digital print-out at 25 microsecond ex. Optical.white cells are counted and sized as RBC Cold agglutinins in high titer tend to give spurious macrocytosis and low RBC count with high MCV and MCHC Low WBC count in leukemic patients.provided machine is properly calibrated and subjected to constabt quality control Elimination of visual fatigue of the medtech EXERCISE 7: Hematocrit Determination Hematocrit PCV. Inadequate mixing of blood cell suspension 4. and Hct. Collection-bad sampling of blood to an adequate flow from skin ouncture o prolonged used of tourniquet o insufficient mixing of venous blood which has settled after collection 2. Presence of yeast cells in the diluting fluids 8. Failure to discard the first few drops before filling the chamber 7. Faulty calibrated pipette 12. Pipetting.Computation of Cell Counts ( ( ) ) Where: Avg = average number of cells/mm3 D (mm) = depth factor DF = dilution factor A (mm3) = area counted Advantages of Manual Counting Do not require expensive reagents and equipments No constant calibration and quality control Less expensive No special laboratory setting needed Automated Hemocytometry 1. Electrical.WBC appears to be fragile and escape being counted Anemic and patients receiving immunosuppressive drugs will give a low WBC count Erroneous PCV occurs if there are sever electrolyte and serum protein abnormalities Advantages of Electronic Cell Counted Over Hemocytometer Generally faster and more précised Human error avoided.inaccurate pipetting of blood and diluting fluid o presence of bubbles 3.Blood cells are counted as deflection of light beams as they pass through darkfield area of the machine which are converted to electrical impulses by a photomultiplier tube ex. Celloscope. Frommer-Sanborn Autocytometer. Careless counting of cells within the chambers 10. Hgb. RBC. Coulter counter.
normally barely visible.6% Sodium oxalate 1. muscular activity.000 rom = 10 min 15. red cell layer.adequate duration and speed 10-12 thousand rpm for 5 minutes.000 WBC/mm3 Plasma Layer. sickle cell anemia Sample: posture. several layer are formed: a.000 rpm = 5 min o radius of centrifugation 22.bottom layer. in the presence of lipidemia. Macrohematocrit Method Anticoagulant Used Wintrode Method Haden's modification Van Allen's Sanford and Magath Bray's Method o o o o o 1. Microhematocrit Method o uses heparinized capillary tubes. Body Hematocrit .thin.5 cm 3000 rpm 30min 15 cm 3500 rpm 30-45 min o length of centrifugation red blood cell count red cell size Sources of Error in Hematocrit Reading Centrifugation. the layer is several mm thick Advantages: less amount of blood needed less error due to less plasma trapped less time involved which makes the method very reliable from the clinical standpoint fairly accurate and gives about 2% variation only when different technologist read the same hematocrit preparation 3. nucleated RBC’s 1 mm buffy coat layer represent about 10. the tubes are centrifuge for 5 minutes and the length of the packed red cell is measured by the hematocrit reader .9% higher than venous blood 2. spherocytosis. Venous Hematocrit ratio of red cells to plasma in venous blood measures the proportion of RBC to plasma in the peripheral blood if anticoagulated blood is used. reticulocytes can be found on the topmost layer b.normally pale yellow and fairly clear excessive hemolysis results in cherry red color jaundice produce a deep yellow color Fatty layer.3% Sodium oxalate Heparin no longer used large amount of blood needed time consuming higher percentage of trapped plasma centrifuge at 2-3 thousand rpm in 30 minutes 2. Automated Hematocrit Method o Coulter Counter o Autoanalyzer o Yellow Springs Instrument Factors Affecting Hematocrit Determination relative centrifugation force o Speed of centrifugation 5000 rpm = 30 min 10. Hgb and cell count not affected Immediately after blood loss Dehydration EXERCISE 8: Hemoglobin Determination Hemoglobinometry an estimation of the mount of hemoglobinin a known amount of blood used as as creening test for disease associated with anemia and following the course of disease in the treatment Methods of Determination: 1. white blood cells 3.Reflects the concentration of the red blood cells.1% Sodium oxalate 1. Buffy Coat Layer. and prolonged tourniquet inadequate blood for fixed amount of EDTA: Hct falsely low due to cell shrinkage. Gravimetric Method Methods of Hematocrit Determination 1. not the red cell mass Types of Hematocrit 1. yellowish white layer which consist of: 1. 1-3% of column is trapped plasma Greater amount of trapped plasma-macrocytic anemia. hypochromic anemia. platelets 2.
is converted into alakaline hematin which is true and relatively stable solution. and the color of glass is matched with a rotating disc of tinted glass varying in thickness and in depth of red color.recommended by ICSH Advantages: o Cyanmethemoglobin pigment is stable in dilute solution o Measures all forms of hemoglobinderivatives except sulfhemoglobin o Certified hemoglobin standards are available o Spectral Curve allows the use of different types of spectrophotometer Principle: The diluents (Drabkin’s solution) contains potassium ferricyanideand potassium cyanide Ferricyanide + hemoglobin= Methemeoglobin Methemoglobin + KCN = stable hemoglobin The resulting solution is then read spectrophotometrically and the equivalent hemoglobin is determined from the calibration curve or table Tube Cyanmethemoglobi Drabski Hemoglobin Labelin n Standards (mL) n Concentratio g Solution n (g/dL) (mL) Blank 1 2 3 4 5 0 1 2 3 4 5 5 5 4 3 2 1 0 4 8 12 16 20 .3467g/ 100 g of Hb or 3.blood is drawn by capillary action between 2 glass plates.048-1. Male= 1.47 mg of iron per 1 gm Hb 4. Alkaline Hematin Methods. This method gives as much as 50% error o 2.057. Gasometric Method o indirect method of hemoglobin determination based on the oxygen content of blood o based on the hemoglobin capacity of 1.053 2. A drop of patients blood is placed in each tube . Dare’s Hemoglobinometer.1N HCl. in the presence of acid like 0. Alkali hematin is also diluted with water drop by drop until the color matches that of the standard solution not ideal for use in infants and children since HbF is resistant to denaturation by alkali Cyanmethemoglobin .056. Chemical Method o indirect method of hemoglobin determination based on the total iron content of blood regarded as bound to hemoglobin o based on the hemoglobin iron content of 0. Patient’s undiluted blood is absorbed into the absorbent pad and the color of blotted blood is compared with the colored scale. Acid hematin is diluted with water drop by drop until the color matches that of the standard 4. Tallquist Method. and oxygen is liberated and measured o used to calibrate instruments for Hgb determination and not for clinical work o correction is done for temperature and pressure o Formula: 3. Female= 1. Electrophoresis 6. Acid Hematin Methods. after which the drop of blood may either sink of float is the specific gravity value. Automated Coulter Counter 5. in the presence of an alkali. The tube wherein the drop of blood remained suspended for 10-12 seconds as they are being coated with copper proteinate. is converted into acid hematin which is a brown solution. Normal range= 1.hemoglobin in known quantity of blood . Gives as much as 30% error 3.hemoglobin in known quantity of blood .34 ml oxygen Van Slyke’s Method (improved method of Haldane and Smith) o determine oxygen and carbon dioxide and is measured by the amount of mmHg displaced o sample is hemolyzed with saponin. Colorimetric Method Direct Visual Colorimetric Method 1.the specific gravity of blood is the ratio of the weight of the volume of blood to the weight of the same volume of water at room temperature o method for screening blood donors Copper Sulfate Method o A set up of tubes containing CuS04 solution of different but known specific gravities is prepared.uses absorbent pads with lithographed color scale representing values ranging from 10-100 %.
increased RDW mixed = increased RDW o mean corpuscular avergae thickness average height/ thickness of RBC MCAT = MCV/ MCD .2 mean corpuscular hemoglobin concentration o average concentration of Hb in the RBC o reference values 31-37 = normochromic > 37 = hyperchromic < 31 = hypochromic o at most 37 g/dL of Hb o ( ) mean corpuscular hemoglobin o average weight of hemoglobin in each RBC o reference values 26. altitude.9-1.1 saturation index average amount of Hemoglobin per unit volume of cell SI = CI/VI or %Hb/%Hct Normal Value: 0.8-1. moderate and high concentration to monitor assay performance Hemoglobin Values are affected by: age.34 = normal o ( ) other indices: o red cell distribution width variation in mean diameter of RBC Normal = 11.o o the standard is used to build a standard covering the concentration range and to prepare quality control samples at low.14% macrocytosis = increased MCV. pregnancy o o Rule of Three 3 x RBC = Hb (g/dL) 3 x Hb = Hct + 3 (%) Red Blood Cell Indices commonly used parameters to assess anemia mean corpuscular volume o average volume of a single RBC in a given blood volume o reference values 80-100 = normal > 100 = macrocytic < 80 = microcytic dimorphic RBC’s yield a normal MCV increased reticulocytes increases MCV o ( ) ( ) o color index amount of Hb in each RBC as compared w/ the average size of a normal RBC CI= % Hb / % RBC Normal value = 0.9-11 volume index average size of RBC as compared w/ the average size of normal RBC VI = % Hct/ % RBC Normal value = 0. sex. normal RDW IDA = decreased MCV.