erythrocyte sedimentation rate(ESR) .pptx

The erythrocyte sedimentation rate(ESR) measures the rate of settling ie. Sedimentation of erythrocytes in millimeters in an anticoagulated Whole blood In a vertical tube at room temperature.
Anticoagulated blood is allowed to stand in a glass tube for 1hr and the length of column of plasma above the red cells is measured in millimeters.
Simple , inexpensive and indirect measure of acute phase reactants.
ESR and CRP are two tests commonly used to screen for inflammation and /or tissue destruction.They are not diagnostic of any disease but indicate need for further studies.

1. ErythroCyte sedimentation rate and packed cell volume Dr.Ankita singh

2. INTRODUCTION Defination- The erythrocyte sedimentation rate(ESR) measures the rate of settling ie. Sedimentation of erythrocytes in millimeters in an anticoagulated Whole blood In a vertical tube at room temperature. Anticoagulated blood is allowed to stand in a glass tube for 1hr and the length of column of plasma above the red cells is measured in millimeters. Simple , inexpensive and indirect measure of acute phase reactants . ESR and CRP are two tests commonly used to screen for inflammation and /or tissue destruction.They are not diagnostic of any disease but indicate need for further studies. ESR is helpful in differentiating functional from organic disease,IDA from anaemia of chronic disease,angina pectoris from MI,Acute appendicitis from other causes of acute abdomen. ESR is important criterion in establishing the diagnosis of temporal arteritis and polymyalgia rheumatics.

3. Stages of ESR Stage1 - Formation of rouleaux or lag phase (10min).Red cells stack together like a pack of coins.ESR mainly depends on this stage. Stage2 -Sinking of rouleaux or decantation phase (40min).Rapid and constant sedimentation.The longer the tube,the longer is this stage and higher the value of ESR.Rouleaux of red cells settle more rapidly. Stage3 -Packing of rouleaux(10min).Slow sedimentation.

4. Factors affecting ESR Factors increasing ESR OLD AGE PREGNANCY ANEMIA. ELEVATED FIBRINOGEN ELEVATED GAMMAGLOBULINS MACROCYTOSIS NEPHROTIC SYNDROME(HYPOALBUMINEMIA) TECHNICAL FACTORS -HIGH TEMPERATURE,TILTING OF TUBE. Factors decreasing ESR MARKED LEUCOCYTOSIS POLYCYTHEMIA LOW FIBRINOGEN,AFIBRINOGENEMIA SICKLE CELL DISEASE, ANISOPOIKILOCYTOSIS, SPHEROCYTOSIS. MICROCYTOSIS TECHNICAL FACTORS -VIBRATION OF TUBE,CLOTTING OF BLOOD,SHORT ESR TUBE.

5. INDICATIONS It is recommended to measure ESR in- infections , inflammation or a neoplastic suspected symptomatic individuals in whom a specific diagnosis has not been established. To monitor disease activity in TB,Rheumatic fever,Hodgkin’s disease,Giant cell arteritis,& Polymyalgia rheumatics. As a diagnostic criterion for temporal arteritis and polymyalgia rheumatica.

6. Methods for estimation Westergreen method Wintrobe method Automated method(sedimat,Ves-matic,ESR stat-plus)

7. Westergreen method High Elevated ESR CAN BE DETECTED BY THIS METHOD BECAUSE OF TALLER COLUMN HEIGHT. EQUIPMENT- WESTERGREN ESR TUBE - THIS IS STRAIGHT GLASS PIPETTE MEASURING 300mm(0-200mm CALIBRATED),THE MARKINGS ARE ONLY OVER THE LOWER 2/3 OF TUBE .THE TUBE IS OPEN ENDED INTERNAL DIAMETER SHOULD NOT BE LESS THAN 2.55mm.TUBE SHOULD BE CLEAN & DRY. WESTERGREN STAND -THIS HOLDS THE TUBE IN A MOTIONLESS,VERTICAL POSITION. ANTICOAGULANT-DILUENT SOLUTION -TRISODIUM CITRATE DIHYDRATE IS ANTICOAGULANT OF CHOICE.ITS COMPOSITION IS TRISODIUM CITRATE DIHYDRATE 32.08gm+DISTILLED WATER1000ml.ITS IS FILTERED THROUGH A STERILE MEMBRANE INTO A STERILE CONATINER AND STORED IN A REFRIGERATOR AT 4°C.IT CAN BE KEPT FOR SEVERAL MONTHS ,BUT IF TURNS TURBID DISCARD IT.

8. ALTERNATE ANTICOAGULANT IS EDTA ( 1.5mg/ml), HOWEVER SUCH ANTICOAGULATED BLOOD SHOULD BE DILUTED WITH TRISODIUM CITRATE DIHYDRATE JUST BEFORE THE TEST(1TRISOD:4BLOOD). IF SPECIMEN IS KEPT AT ROOM TEMPERATURE,TEST SHOULD BE CARRIED OUT WITHIN 4hrs AND IF STORED AT4°C THEN WITHIN 6hrs. BLOOD ANTICOAGULATED WITH EDTA CAN BE TESTED WITHIN 24hrs IF STORED AT 4°C(PROVIDED IT IS DILUTED WITH TRISODIUM CITRATE BEFORE TESTING) AND BROUGHT TO ROOM TEMPERATURE.

9. Method MIX ANTICOAGULATED BLOOD SAMPLE THOROUGHLY.BLOOD IS FILLED UPTO ‘0’MARK.A RUBBER BULB OR MECHANICAL DEVICE SHOULD BE USED FOR FILLING. THERE SHOULD BE NO AIR BUBBLES IN BLOOD. THE TUBE IS PLACED IN A STRICTLY VERTICAL POSITION IN THE ESR STAND AND LEFT UNDISTURBED FOR 1hr.AVOID KEEPING IN DIRECT SUNLIGHT AND SHOULD NOT BE SUBJECTED TO VIBRATIONS. AFTER EXACTLY 1hr,READ THE HEIGHT OF THE COLUMN OF PLASMA ABOVE THE RED CELL COLUMN IN mm.BUFFY COAT LAYER SHOULD NOT BE INCLUDED. RESULT IS EXPRESSED IN __mm in 1hr.

10. Reference range Males10-50YEARS-:<10mm in 1hr Males51-60years-:<12mm in 1hr Males61-70years-:<14mm in 1hr Males>70years-:<30mm in 1hr Females17-50years-:<12mm in 1 hr
Females51-60years-:<19mm in 1hr Females61-70years-:<20mm in 1hr Females>70-:<35mm in 1hr NEWBORNS-:0-2mm in 1hr Children to puberty-:0-13mm in 1hr

11. Precautions Use The Correct Proportions Of Blood and ANTICOAGULANT.MIX BLOOD AND ANTICOAGULANT THOROUGHLY.THERE SHOULD BE NO CLOTS AND AIR BUBBLES. SIMPLE FORMULAS FOR MAXIMUM UPPER LIMIT OF ESR ACCORDING TO GENDER ARE MALES:AGE IN YEARS/2, FEMALES:AGE IN YEARS +10/2 THE ABOVE REFERENCE RANGE RELATES TO TEST PERFORMED AT ROOM TEMPERATURE 18-25°C .IF TEMPERATURE IS HIGH ESR INCREASES. ESR TUBE MUST BE KEPT VERTICAL,EVEN A SLIGHT TILT WILL INCREASE ESR.

12. Wintrobes method WINTROBE TUBE CAN BE USED FOR ESTIMATION OF BOTH ESR AND PCV.AFTER OBTAINING ESR IN THE FIRST HOUR,THE TUBE CAN BE SPUN IN A CENTRIFUGE TO GET PCV. THIS METHOD IS MORE RELIABLE WHEN ESR IS LOW,WHILE WESTERGREN IS MORE SENSITIVE FOR HIGH ESR EDTA OR DOUBLE OXALATE IS USED AS AN ANTICOAGULANT. WINTROBE TUBE IS SHORTER110mm & INTERNAL DIAMETER IS 3mm. REFERENCE RANGE IS MALES:0-9mm in 1hr, FEMALES:0-20mm in 1hr,CHILDREN :0-13m in 1hr.

13. CORRECTED ESR - Find the horizontal lines which represent ESR FOR 1 ST HOUR -FOLLOW THIS ACROSS CHART TILL IT INTERSECTS THE VERTICAL LINE. -VERTICAL LINE REPRESENT BLOOD CELL VOLUME . -FOLLOW NEAREST CURVE LINE TILL IT INTERSECT HEAVY LINE AT 1)42% per 100ml Female 2)47%per 100ml Male -At point of intersection the value on horizontal lines for corrected ESR

14. Packed cell volume IT IS THE VOLUME OCCUPIED BY THE RED CELLS WHEN A SAMPLE OF ANTICOAGULATED BLOOD IS CENTRIFUGED. IT INDICATES AS HAEMATOCRIT OR ERYTHROCYTE VOLUME FRACTION. IT IS EXPRESSED AS A PERCENTAGE OF ORIGINAL VOLUME OF BLOOD OR AS A DECIMAL FRACTION. PCV SHOULD BE USED FOR TRADITIONAL TECHNIQUE BY CENTRIFUGATION WHILE HEAMATOCRIT FOR AUTOMATED HAEMATOLOGY ANALYZERS . REFERENCE RANGE -ADULT MALES:40-50%, ADULT FEMALES:38-45%(NON PREGNANT), PREGNANT FEMALES:36-42%, CHILDREN 6-12YEARS:37-46%, CHILDREN 6MONTHS-6YEARS:36-42%, INFANTS 2-6MONTHS:32-42%, NEWBORNS:44-60%

15. Uses DETECTION OF PRESENCE OR ABSENCE OF ANEMIA OR POLYCYTHEMIA. ESTIMATION OF RED CELL INDICES(MEAN CELL VOLUME AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION). CHECKING ACCURACY OF HAEMOGLOBIN VALUE (HB in GM’s/do*3=PCV)

16. Methods THERE ARE TWO METHODS FOR ESTIMATION MACROMETHOD :-WINTROBE METHOD MICROMETHOD :-MICROHEMATOCRIT METHOD.THIS METHOD IS PREFERRED BECAUSE ITS RAPID, CONVENIENT,REQUIRES ONLY A SMALL AMOUNT OF BLOOD,CAPILLARY BLOOD FROM SKIN PUNCTURE CAN BE USED AND A LARGE NUMBER OF SAMPLES CAN BE TESTED AT ONE TIME.THIS METHOD IS MORE ACCURATE AS PLASMA TRAPPING IN RED CELL COLUMN IS LESS.

17. MACROMETHOD (WINTROBE method) PRINCIPLE -ANTICOAGULATED WHOLE BLOOD IS CENTRIFUGED IN A WINTROBE TUBE TO COMPLETELY PACK THE RED CELLS.IT IS SLOW AND INCONVENIENT FOR ROUTINE. BOTH ESR AND PCV CAN BE MEASURED. EQUIPMENT -WINTROBE TUBE :-THIS TUBE IS ABOUT 110mm IN LENGTH AND HAS 100 MARKINGS ,EACH AT THE INTERVAL OF 1mm.INTERNAL DIAMETER IS 3mm.IT CAN HOLD ABOUT 3ml OF BLOOD. PASTEUR PIPETTE WITH A RUBBER BULB AND A SUFFICIENT LENGTH OF CAPILLARY TO REACH THE BOTTOM OF WINTROBE TUBE. CENTRIFUGE WITH A SPEED OF 2300G. SPECIMEN :-VENOUS BLOOD COLLECTED IN EDTA(1.5mg EDTA FOR 1ml OF BLOOD) OR IN DOUBLE OXALATE.TEST SHOULD BE PERFORMED WITHIN 6hrs OF COLLECTION.

18. Method MIX THE ANTICOAGULATED BLOOD SAMPLE THOROUGHLY. DRAW THE BLOOD SAMPLE IN A PASTEUR PIPETTE AND INTRODUCE THE PIPETTE UP TO BOTTOM OF WINTROBE TUBE. FILL THE TUBE FROM THE BOTTOM EXACTLY UPTO THE 100MARK(1ml of blood) DURING FILLING ,TIP OF PIPETTE IS RAISED,BUT SHOULD REMAIN UNDER THE RISING MENISCUS TO AVOID FOAMING. CENTRIFUGE THE SAMPLE AT 2300g FOR 30min. TAKE THE READING OF THE LENGTH OF THE VOLUME OF RED CELLS. HAEMATOCRIT CAN BE EXPRESSED EITHER AS PERCENTAGE OR AS A FRACTION OF THE TOTAL VOLUME OF BLOOD SAMPLE .

19. SIGNIFICANCE IN ANAEMIA,PCV IS BELOW THE LOWER LEVEL OF NORMAL RANGE. PCV IS RAISED IN DEHYDRATION ,SHOCK,BURNS& POLYCYTHEMIA. AFTER CENTRIFUGATION OF ANTICOAGULATED BLOOD ,THREE ZONES CAN BE DISTINGUISHED IN WINTROBE TUBE FROM ABOVE DOWNWARDS-PLASMA,BUFFY COAT(A SMALL GREYISH LAYER OF WHITE CELLS AND PLATELETS ABOUT 1mm THICK) AMD PACKED RED CELLS. NORMAL PLASMA IS STRAW COLOURED.IT IS COLOURLESS IN IDA,PINK IN HEMOLYSIS/HAEMOGLOBINEMIA, YELLOW IN JAUNDICE & IN HYPERTRIGLYCERIDEMIA MILKY. INCREASED THICKNESS OF BUFFY COAT LAYER IF WHITE CELLS OR PLATELETS ARE INCREASED. SMEARS CAN BE MADE FROM BUFFY COAT IN LUPUS ERYTHEMATOUS CELLS(LE),MALARIA PARASITE OR IMMATURE CELLS.

20. Micromethod PRINCIPLE :-ANTICOAGULANT WHOLE BLOOD IS CENTRIFUGED IN A CAPILLARY TUBE OF UNIFORM BORE TO PACK THE RED CELLS.CENTRIFUGATION IS CARRIED IN A SPECIAL MICROHEMATOCRIT. CENTRIFUGE TILL PACKING OF RED CELLS IS AS COMPLETE AS POSSIBLE .THE READING (LENGTH OF PACKED RED CELLS AND TOTAL LENGTH OF THE COLUMN )IS TAKEN USING A MICROHEMATOCRIT READER,A RULER,OR ARITHMETIC GRAPH PAPER.

21. EQUIPMENT MICROHEMATOCRIT CENTRIFUGE :-IT SHOULD PROVIDE RELATIVE CENTRIFUGAL FORCE OF 10,000-15,000g FOR 5MIN. CAPILLARY HEMATOCRIT TUBES :-THESE ARE DISPOSABLE GLASS TUBES 75mm IN LENGTH AND 1.2mm IN INTERNAL DIAMETER.THEY ARE OF TWO TYPES PLAIN (NO ANTICOAGULANT)& HEPARINIZED (COATED WITH DRIED FILM OF 2UNITS OF HEPARIN). FOR PLAIN TUBES , ANTICOAGULATED VENOUS BLOOD IS NEEDED.HEPARINIZED TUBES ARE USED FOR BLOOD OBTAINED FROM SKIN PUNCTURE. PLASTIC TUBES (POLYCARBONATE) ARE SAFER THAN GLASS TUBES AS NO RISK OF TRANSMISSION . TUBE SEALANT LIKE PLASTIC SEALANT OR MODELLING CLAY OR SPIRIT LAMP FOR HEAT SEALING SHOULD BE USED. MICROHEMATOCRIT READER OR RULER OR ARITHMETIC GRAPH PAPER SHOULD BE USED.

22. Method FILL THE CAPILLARY TUBE BY APPLYING ITS TIP TO THE BLOOD (EITHER FROM SKIN PUNCTURE OR ANTICOAGULATED VENOUS BLOOD, DEPENDING ON THE TYPE OF TUBE USED).ABOUT 2/3TO 3/4LENGTH OF THE CAPILLARY TUBE SHOULD BE FILLED WITH BLOOD(ABOUT 15mm SHOULD BE LEFT UNFILLED). SEAL THE OTHER END OF CAPILLARY TUBE (WHICH WAS NOT IN CONTACT WITH BLOOD)WITH MODELING CLAY OR A PLASTIC SEALANT.IF IT IS NOT AVAILABLE ,HEAT SEAL THE TUBE USING A SPIRIT LAMP. THE FILLED TUBES ARE PLACED IN RADIAL GROOVES OF THE CENTRIFUGE WITH THE SEALED ENDS TOWARD THE OUTER RIM GASKET.COUNTERBALANCE BY PLACING THE TUBES IN THE GROOVES OPPOSITE TO EACH OTHER. CENTRIFUGE AT RELATIVE CENTRIFUGAL FORCE 12,000g FOR5-10min TO COMPLETELY PACK THE RED CELLS. IMMEDIATELY REMOVE THE TUBES FROM THE CENTRIFUGE AND STAND THEM UPRIGHT.THE TUBE WILL SHOW 3LAYERS TOP TO BOTTOM -COLUMN OF PLASMA,THIN LAYER OF BUFFY COAT,AND COLUMN OR RED CELLS.

23. WITH THE HAEMATOCRIT READER, HEMATOCRIT IS DIRECTLY READ FROM THE SCALE.IF READER IS NOT AVAILABLE ,THE TUBE IS HELD AGAINST A RULER AND THE HAEMATOCRIT IS OBTAINED BY LENGTH OF RED CELL COLUMN IN mm/LENGTH OF TOTAL COLUMN IN mm. TO OBTAIN PCV ,THE ABOVE RESULT IS MULTIPLIED BY100. DISADVANTAGE :-RISK OF BREAKAGE OF GLASS, DIFFICULTY IN READING THE CORRECT LEVEL DUE TO SMALLNESS OF TUBE,TUBE MAY HAVE UNEVEN BORE, VARIABILITY OF PLASMA TRAPPING,AND UNEVEN SURFACE PRODUCED DUE TO SEALING.

24. BIBLIOGRAPHY Essentials of clinical pathology. Dacie and Lewis practical hematology. Harsh and Mohan practical hematology.