The document discusses the erythrocyte sedimentation rate (ESR), including its definition, history, factors that influence it, normal values, and methods of measurement such as the Westergren and Wintrobe methods. It also describes the zeta sedimentation ratio (ZSR) and its advantages over traditional ESR measurement, such as being unaffected by anemia. The clinical significance of ESR is that it can help follow the course of disease, establish prognosis, and support diagnoses, though it is a non-specific test.

1. ERYTHROCYTE SEDIMENTATION RATE WITH SPECIAL REFERENCE TO ZSR & METHOD OF ESR IN INFANTS MODERATOR- Dr SANJEEV NARANG

2. ERYTHROCYTE SEDIMENTATION RATE DEFINITION The Length of fall of the top of the column of erythrocytes in a given interval of time is called ERYTHROCYTE SEDIMENTATION RATE

3. HISTORY THE ESR TEST WAS INVENTED IN 1897 BY POLISH DOCTOR EDMUND BIERNACKI . IN 1918 SWEDISH PATHOLOGIST ROBERT SANNO FAHRAEUS DECLARED THE SAME & ALONG WITH ALF VILHELM WESTERGREN ARE EPONYMOUSLY REMEMBERED FOR FAHRAEUS-WESTERGREN TEST.

4. TERMS ACUTE PHASE REACTANT –A substance in the blood that increases as a response to an acute condition such as INFECTION,INJURY,BURNS etc. ERYTHROCYTE SEDIMENTATION RATE (ESR) –The distance that RBC’s settle in a tube of blood in one hour. ROULEAUX – The stacking of RBC’s ,caused by extra or abnormal proteins in the blood that decrease the normal distance red cells maintain between each other.

5. FACTORS INFLUENCING E.S.R FACTORS THAT INCREASE E.S.R OLD AGE FEMALE PREGNANCY ANEMIA R.B.C ABNORMALITIES MACROCYTOSIS TECHNICAL FACTORS DILUTION PROBLEMS INCREASED TEMP. OF SPECIMEN TILTED E.S.R TUBE ELEVATED FIBRINOGEN LEVELS INFECTION INFLAMMATION MALIGNANCY FACTORS THAT DECREASES ESR EXTREME LEUKOCYTOSIS POLYCYTHEMIA RBC ABNORMALITIES SPHEROCYTOSIS ACANTHOCYTOSIS MICROCYTOSIS TECHNICAL FACTORS DILUTION PROBLEMS INADEQUATE MIXING CLOTTED BLOOD SAMPLE SHORT ESR TUBE VIBRATION DURING TESTING PROTEIN ABNORMALITIES HYPOFIBRINOGENEMIA HYPOGAMMAGLOBULINEMIA DYSPROTEINEMIA

6. STAGES OF ESR Initial 10 minutes –little sedimentation as Rouleaux formation –STAGE OF ROULEAUX Next 40 minutes –settling occurs at constant rate –STAGE OF SEDIMENTATION Final 10 minutes – sedimentation slows as cell pack at bottom of tube

7. WESTERGREN’S METHOD Reference method. 30 cm long 2.5 mm internal diameter. Calibrated from 0 to 200. Capacity 1 ml. Rack is used with levelers as needed for vertical tube. REAGENT – 0.105 M (0.10 – 0.156 ) of sodium citrate is used as a anticoagulant – diluent.

8. PROCEDURE-WESTERGREN Add 2 ml of whole blood to 0.5 ml of 3.8% sodium citrate & mix. Fill pipette up to zero mark. Place pipette exactly in vertical position at room temperature. After 1 hour the distance from 0 mark to top of RBC column is recorded as ESR,

9. MODIFIED WESTERGREN’S EDTA Instead of citrate as anticoagulant. 2 ml of EDTA diluted with 0.5 ml of 3.8 % sodium citrate or 0.5 ml of 0.85 % sodium chloride. Undiluted EDTA blood gives poor precision. DILUTED BLOOD = ( UNDILUTED ESR *0.86 ) -12.

10. ESR –NORMAL VALUES (WESTERGREN’S ) AGE (YEARS) 95%UPPER LIMITS(MM IN HOUR) MEN 17 – 50 10 51 – 60 12 61 – 70 14 >70 ABOUT 30 WOMEN 17 – 50 12 51 – 60 19 61 – 70 20 >70 ABOUT 35 PREGNANCY First half 48 (62 if anemia) Second half 70 (95 if anemia)

11. WINTROBE’S METHOD Wintrobe’s tube Length = 110 mm. Internal Diameter 3.0 mm Two markings . 1st marking for ESR (ABOVE DOWNWARDS ) 2 nd marking for PCV (BELOW UPWARDS ) GRADUATION FROM 0 TO 100 Wintrobe’s tube stand. Pasteur pipette Timer

12. PROCEDURE Mix blood carefully with EDTA anticoagulant. Fill tube up to zero mark. Place the tube exactly in vertical position in stand. Set timer for one hour. At the end of one hour note the level of erythrocyte column in mm after one hour.

13. WINTROBE’S METHOD

14. PRECAUTIONS Wash the tubes under running water using a thick wire. Dry the tube in incubator. In case of infants & if blood is insufficient use LANDAU METHOD.

15. NORMAL VALUES BY WINTROBE METHOD MALE = 0-9 MM AFTER 1 st HOUR FEMALE = 0-20 MM AFTER 1 st HOUR

16. MICRO SEDIMENTATION METHOD (LANDAU ) REQUIREMENT. FASTING CAPILLARY BLOOD (FINGER TIP , HEEL OR TOE ). 5.0 g / dl sodium citrate solution. LANDAU PIPETTE (GRADUATED FROM 0 TO 50 MM ). LANDAU Pipette stand. Suction device for drawing blood into pipette. Capillaries for blood collection.

17. PROCEDURE. Attach landau pipette to suction device. Draw 5.0 g/dl sodium citrate up to 1st line of stem. Draw blood by suction device up to 2 nd mark. Draw citrate / blood into bulb & mix thoroughly. Force back the mixture into stem of pipette. Set the upper level to 0 mark at top. Place it in vertical stand. Note reading after one hour.

18. NORMAL VALUES MALE = 0-5 MM AFTER 1 st HOUR FEMALE = 0-8 MM AFTER 1 st HOUR

19. CHILDREN NEWBORN 0 TO 2 MM/HR NEONATAL TO PUBERTY 3 TO 13 MM/HR 8 DAYS AFTER DELIVERY ABOUT 4MM/HR 14 DAYS AFTER DELIVERY ABOUT17MM/HR

20. SOURCES OF ERROR Proper anticoagulant -1)right volume. -2)rightconcentration. Bubbles left in tube. Hemolysis. Un cleanliness of tube. Tilting of tube. Altered temperature. Time after collection .

21. VES MATIC 20 INSTRUMENT AUTOMATED BENCH TOP ANALYSER TO MEASURE ESR. BLOOD COLLECTED IN CUVETS. SAMPLE IS LEFT TO SEDIMENT. 18 DEGREE SLANTING OF TUBES CAUSES ACCLERATION OF ESR. RESULTS COMPAREABLE TO 1)WESTERGREN S 1 st HOUR TO BE OBTAINED IN 25 MINUTES. 2)WESTERGREN S 2 nd HOUR TO BE OBTAINED IN 45 MINUTES.

22. ZETA POTENTIAL THE ZETA POTENTIAL RESULTS FROM NEGATIVELY CHARGED SIALIC ACID GROUPS ON THE RED CELL MEMBRANE. FIBRINOGEN & GAMMA GLOBULINS DECREASE ZETA POTENTIAL. DECREASED ZETA POTENTIAL RESULTS IN INCREASED ROULEAUX FORMATION. THIS RESULTS IN INCREASED ESR. THIS MECHANISM IS MOST LIKELY EXPLANATION FOR INCREASED ESR IN DISEASE.

23. ZETA SEDIMENTATION RATIO DEFINITION - IT IS THE MEASUREMENT OF THE EASE WITH WHICH RBC’S WILL PACK UNDER A STANDARDISED COMPACTION – DISPERSION STRESS. IT IS INVERSELY RELATED TO ZETA POTENTIAL OF THESE CELLS WHEN SUSPENDED IN PARTICULAR PLASMA UNDER CONSIDERATION.

24. ZETA SEDIMENTATION RATIO .REQUIRES A ZETAFUGE. ZETAFUGE IS A CENTRIFUGE WHICH REQUIRES SPIN CAPILLARY TUBES IN VERTICAL POSITION IN FOUR 45 SECONDS CYCLES. ROULEAUX FORMATION & SEDIMENTATION IN JUST 3 MINUTES. READING IS KNOWN AS ZETACRIT. ZETA SEDIMENTATION RATIO = TRUE Hct / ZETACRIT X 100. NORMAL VALUES = 41 TO 54 % FOR BOTH SEXES.

25. PROCEDURE A BLOOD SAMPLE OF 100 ul CONTAINED IN A VERTICAL TUBE IS SPUN AT 400 rpm FOR 45 SECONDS. THE RESULTANT 7-8 g FORCE CAUSES RBC’S TO TRAVEL OUTWARD UNTIL THEY APPROACH THE OUTER EDGE OF THE TUBE. THE CAPILLARY TUBES ARE ROTATED 180 DEGREES & CENTRIFUGE IS RESTARTED. AS CENTRIFUGE HEAD REGAINS IT’S OPERATING SPEED THE CLUMPED RBC’S NOW TRAVEL FROM INNER WALL OF TUBE . THEY ARE PARTIALLY DISPERSED DURING THE INITIAL PHASES OF THIS JOURNEY BEFORE BEING DEPOSITED ONCE MORE IN OUTER WALL.

26. PROCEDURE IN THE PROCESS THEY ARE EXPOSED TO NORMAL DOWNWARD FORCE OF GRAVITY AND THUS COME TO REST UPON OUTER EDGE OF TUBE AT POINT SOMEWHAT LOWER THAN STARTING PHASE. THIS PROCESS IS REPEATED 4 TIMES . THIS CAUSES RBC’S TO FOLLOW ZIG ZAG PATH DOWN THE CAPILLARY TUBE & BECOMES MORE DENSELY PACKED. FOUR 45 SEC. CYCLES OF COMPACTION ,DISPERSION & RECOMPACTION ARE EMPLOYED . AT THE CONCLUSION OF THIS 3 MINUTE PERIOD,THE DEGREE OF COMPACTION ACHIEVED BY RBC’S ARE MEASURED.

27. MEASUREMENT OF ZSR TO MEASURE ZSR A CAPILLARY TUBE IS READ AFTER THE COMPACTION – DISPERSION CYCLES . ZSR CORRESPONDS WITH UPPER MENISCUS OF THE RED CELL COLUMN . THE MENISCUS OF ZETACRIT IS NOT COMPLETELY HORIZONTAL. THE TAIL OF RED CELLS RISES ABOVE IT ON ONE SIDE & A SMALL WEDGE OF PLASMA DEPRESSES IT ON THE OTHER. ZETACRIT IS MARKED AT YHE “ KNEE “ OF THE CURVE BETWEEN THE RED CELLS & THE MENISCUS .

28. ADVANTAGES OF ZSR CAPILLARY TUBES ARE EASIER TO FILL. REQUIRES LESS SAMPLE. CAPILLARY TUBES ARE CHEAPER THAN STANDARD ESR TUBES. ZSR IS UNAFFECTED BY ANEMIA .

29. DRAWBACKS OF ZSR RESULTS ARE EXPRESSED IN NEW UNITS i.e. % RATHER THAN IN MM / HOUR. NEED TO PURCHASE SPECIAL CAPILLARY TUBES & A SPECIFIC NEW INSTRUMENT CALLED ZETAFUGE .

30. APPLICATIONS OF ZSR 1- IN THOSE SITUATIONS WHERE ESR IS ALREADY USED. 2-ANEMIC PATIENTS. 3-MULTIPHASIC HEALTH SCREENING CENTRES. 4-BLOOD BANK DONORS.

31. ZETAFUGE

32. DETERMINATION OF ZSR

33. VALUES OF ZSR NORMAL 51 TO 54 % MILDLY ELEVATED 55 TO 59 % MODERATELY ELEVATED 60 TO 64 % MARKEDLY ELEVATED >65 %

34. CORRECTED ESR TO ELIMINATE THE INFLUENCE OF ANEMIA ON ESR. CORRECTED ACCORDING TO VOLUME OF CELLS .

35. CORRECTION FOR ESR FIND THE HORIZONTAL LINE WHICH REPRESENT ESR FOR 1st hour. FOLLOW THIS ACROSS CHART TILL IT INTERSECTS THE VERTICAL LINE. VERTICAL LINE REPRESENTS BLOOD CELL VOLUME. FOLLOW NEAREST CURVED LINE TILL IT INTERSECTS HEAVY LINE AT 1) 42 % PER 100 ml > FEMALE 2) 47 % PER 100 ml > MALE. AT POINT OF INTERSECTION READ THE VALUE ON HORIZONTAL LINE FOR CORRECTED ESR.

36. GRAPH FOR CORRECTED ESR

37. CLINICAL SIGNIFICANCE OF ESR. TO FOLLOW THE COURSE OF DISEASE. TO ESTABLISH PROGNOSIS IN CERTAIN CHRONIC DISEASES. INCREASE IN TWO OR MORE CONSECUTIVE TESTS INDICATES CONTINUATION OF INCREASED ACTIVITY. TO DISTINGUISH BETWEEN ORGANIC/NON ORGANIC DISORDERS. TO SUPPORT THE DIAGNOSIS.

38. LIMITATIONS OF ESR ESR IS A NON SPECIFIC PHENOMENON & REFLECTS ONLY CHANGE IN PLASMA PROTEINS PATTERN & VARIATION IN RED CELL COUNT. CANNOT BE USED AS A DIAGNOSTIC TOOL. DOES NOT INDICATE THE NATURE OF DISEASE.

39. REFERENCES HENRY’S CLINICAL DIAGNOSIS &MANAGEMENT BY LAB. METHODS 21/e DACIE & LEWIS PRACTICAL HEMATOLOGY 10 /e T.B. OF MEDICAL LAB TECHNOLOGY-GODKAR 2/e VARIOUS INTERNET SITES

40. THANKS BY- Dr RAVI JAIN