HEMOGLOBIN ESTIMATION AND STAINING OF PERIPHERAL BLOOD SMEAR.pptx

Estimation of hemoglobin and how to stain a peripheral blood smear.
Presented for BDS and MD/MS (Ayu) tutorials.

1. HEMOGLOBIN ESTIMATION AND STAINING OF PERIPHERAL BLOOD SMEAR Dr Atreyee Chakrabarty Junior Resident Department of Pathology, IMS BHU

2. HEMOGLOBIN ESTIMATION

3. INTRODUCTION Hemoglobin is a CHROMOPROTEIN. (gives red color to the whole blood) Major component of RBC cytoplasm - 90% of dry weight of mature RBC - 32% of wet weight of mature RBC WHAT IF HEMOGLOBIN WAS PRESENT IN THE PLASMA? There would be an increase in viscosity of the blood leading to increased blood pressure. There would be an increase in osmotic pressure leading to increased fluid exchange Secretion in urine Rapid destruction by reticuloendothelial system.

4. SYNTHESIS OF HEMOGLOBIN Begins in: PROERYTHROBLAST STAGE First appears in: INTERMEDIATE NORMOBLAST STAGE Continues till: RETICULOCYTE STAGE EARLY NORMOBLAST INTERMEDIATE NORMOBLAST LATE NORMOBLAST

5. Derived from physiological by action of acids, alkalis, oxidizing or reducing agents Loose and reversible binding of oxygen with iron atom. Each molecule of Hb carries 4 molecules of oxygen. Formed by release of oxygen from hemoglobin. Carbon dioxide is attached to the globin part of Hb . Affinity of Hb for carbon dioxide is 20 times that of oxygen. Carbon monoxide binds to sites of oxygen binding in Hb . Affinity for carbon monoxide is 200-300 times that of oxygen. Causes: Drugs Oxidising agents Ferrous iron converted to ferric iron Gives dusky appearance to skin Causes: Drugs ( Sulphonamides ) Chemicals Irreversible - Action of cyanide on Hb .

6. GOWER 1 HB: ζ 2 ε 2 GOWER 2 HB: α 2 ε 2 HB PORTLAND: ζ 2 γ 2 HBF: α 2 γ 2 After 8th week of development γ chain differs from β in 37 amino acids Lesser affinity to 2,3 DPG- so accepts more oxygen from maternal blood 80% of total Hb at birth Disappears by 5 th month VARIANTS OF HEMOGLOBIN Different structural forms of hemoglobin that vary in the structure of the polypeptide chains. Identified by hemoglobin electrophoresis HBA: α 2 β 2 HBA2: α 2 δ 2 Seen in sickle cell anemia Glutamic acid is replaced by valine in the 6 th position in β chain Seen in African Americans Glutamate is replaced by lysine in the 6th position Glutamate is replaced by glutamine in the 12 th position HbE HbI HbJ HbH Barts ’

7. PATHOGENESIS OF SICKLE CELL ANEMIA

8. HEMOGLOBIN LEVELS AGE GROUP HEMOGLOBIN LEVELS (in gm /dl) AT BIRTH 13.6-19.6 2-6 MONTHS 9.5-14.0 6 MONTHS-6 YEARS 11.0-14.0 6 YEARS- 12 YEARS 11.5-15.5 ADULT FEMALE Pregnant 11.0-14.0 Non Pregnant 12.0-15.0 ADULT MALE 13.0-17.0 WHY ARE HEMOGLOBIN LEVELS LOWER IN FEMALES? NOT DUE TO MENSTRUAL LOSSES (20-30 ML) Estrogen has inhibitory effect on erythropoietin Androgen has stimulatory effect on erythropoietin WHY ARE HEMOGLOBIN LEVELS HIGHER IN NEWBORNS? Increased RBC count Relative hypoxia in infants acts as a potent stimulus for erythropoietin secretion

9. INDICATIONS OF HEMOGLOBIN ESTIMATION Anemia refers to reduced hemoglobin or reduced oxygen carrying capacity of blood. Anemia is best assessed by: Hemoglobin estimation Packed cell volume estimation According to severity, anemia can be classified into: Mild: >10 gm /dl but less than lower side normal range Moderate: 7-10 gm /dl Severe: <7 gm /dl Refers to hemoglobin levels above normal Classification: PRIMARY: Total red cell mass increased, erythropoietin normal eg : POLYCYTHEMIA VERA SECONDARY: Total red cell mass increased, erythropoietin increased Eg : HYPOXIA RELATIVE: Plasma volume reduced,total red cell mass normal Eg : DEHYDRATION Along with Packed Cell Volume (PCV) and RBC count  

10. METHODS OF HEMOGLOBIN ESTIMATION Color comparison is done between standard and test sample. VISUAL Tallqvist Sahli’s acid hematin method WHO Hemoglobin color scale PHOTO ELECTRIC Cyanmethemoglobin Oxyhemoglobin Alkali hematin method Measures oxygen carrying capacity Apparatus: VAN SLYKE 1 gram Hb contains 1.34 ml of oxygen Only measures oxyHb and reduced Hb Does not measure carboxyHb , sulfHb or metHb Time consuming Expensive Iron content of hemoglobin is estimated. 100 grams of hemoglobin contains 374 mg iron. Tedious Time consuming Rapid and simple method Used for selection of blood donors It is done by CuSO4 method Gives an idea about cell count and hemoglobin concentration Hemolysing agent causes release of hemoglobin. Potassium ferricyanide and cyanide are added to this forming cyanmethemoglobin . Hb is analysed by spectrophotometry,

11. TALLQVIST CHART Contains a series of lithographed colors. Corresponds to Hb levels between 10-100% Cheap and simple method. Margin of error=20-50% Method is currently obsolete.

12. SAHLI’S HEMOGLOBINOMETER METHOD PRINCIPLE: EQUIPMENT: REAGENTS: DISTILLED WATER 0.1 N HCl OTHERS: STERILE LANCET STERILE GAUZE COTTON SWABS 70% ALCOHOL

13. PARTS COMPARATOR BOX Rectangular plastic box Slot in the middle to accommodate the calibrated hemoglobin tube Non fading, standardised , golden brown glass rods fitted on each side of the slot matching the color. Opaque white glass fitted behind the slot to provide uniform illumination during direct visual color matching. HEMOGLOBIN TUBE Square or round glass tube is calibrated in two colors: Yellow color: g Hb % (preferred) Red color: % Hb Brush is also provided to clean the tube. HEMOGLOBIN PIPETTE Glass capillary pipette Single calibration mark: 20 μ L No bulb because there is no dilution of blood STIRRER Thin glass rod with flattened end Use: Stirring and mixing the blood and dilute acid PASTEUR PIPETTE 8 to 10” glass tube Drawn to a long thin nozzle with a rubber teat Acts as a dropper

14. PROCEDURE

15. QUESTIONS! WHAT IS NORMALITY? WHAT IS N/10 HCL? HOW TO PREPARE IT? CAN WE USE STRONG ACIDS OR ALKALIS INSTEAD OF N/10 HCL? WHY? WHAT IF WE TAKE LESS THAN 8-10 DROPS OF N/10 HCL? WHAT IF WE TAKE MORE THAN 8-10 DROPS OF N/10 HCL? WHY DO WE DISCARD THE FIRST TWO DROPS? WHY DO WE HAVE TO WAIT FOR 6-8 MINUTES? CAN WE USE TAP WATER FOR DILUTING INSTEAD OF DISTILLED WATER? CAN WE USE N/10 HCL FOR DILUTING INSTEAD OF DISTILLED WATER?

16. WHAT IS NORMALITY? - No of gram equivalents in 1 LITRE of water. WHAT IS N/10 HCL? HOW TO PREPARE IT? 1 N HCl = 1+35 g=36 g of HCl in 1 litre of water So N/10 HCl is 36 g of HCl in 10 litres of water or 3.6 gm HCl in 1 litre of water. So basically diluting a 1 N solution 10 times will give us N/10 HCl . CAN WE USE STRONG ACIDS OR ALKALIS INSTEAD OF N/10 HCL? WHY? - NO. Because strong acids and alkalis will cause disruption of hemoglobin. WHAT IF WE TAKE LESS THAN 8-10 DROPS OF N/10 HCL? 1. Blood may not mix well and may clot. 2. All of the Hb will not be converted to acid hematin . So this will lead to a false low value. WHAT IF WE TAKE MORE THAN 8-10 DROPS OF N/10 HCL? The final color that would develop would be much lighter than the actual. This would give a false high value. CAN WE USE TAP WATER FOR DILUTING INSTEAD OF DISTILLED WATER? No. Acid hematin is already not a true solution. Hence, there is a risk of turbidity. Using tap water which has salt, would further increase the chance of turbidity. CAN WE USE N/10 HCL FOR DILUTING INSTEAD OF DISTILLED WATER? Yes. All the Hb would have already been converted to acid hematin , so adding N/10 HCl would not further deepen the color.

17. CAN WE USE N/10 HCL FOR DILUTING INSTEAD OF DISTILLED WATER? Yes. All the Hb would have already been converted to acid hematin , so adding N/10 HCl would not further deepen the color. WHY DO WE HAVE TO WAIT FOR 6-8 MINUTES? To give time for the acid hematin to form. WHY DO WE DISCARD THE FIRST TWO DROPS? To prevent contaminants in the blood that may come from the skin surface. Initial drops might be diluted with tissue fluid and can give a false estimation of Hb .

18. ADVANTAGES DISADVANTAGES Simple Quick Accurate Inexpensive Can be used for mass surveys Acid hematin is not in a true solution. So there may be turbidity. Only measures oxyhemoglobin and reduced hemoglobin. CarboxyHb and MetHb are not estimated. SOURCES OF ERROR PERSONAL Not taking exact 20 cu.mm Not giving enough time for acid hematin to form Using old comparator with faded glass rods TECHNICAL - Color matching is done visually so it becomes subjective PRECAUTIONS Donot squeeze finger while collecting blood. No blood should be collected that sticks outside the pipette tip. Only recommended time should be allowed for each step. Avoid overdilution because color cannot be concentrated once it becomes lighter than the color in the comparator. There should be a uniform golden brown color throughout the solution. Dark color near the bottom of the tube indicates improper mixing. Take 3 readings to reduce personal error.

19. WHO HEMOGLOBIN COLOR SCALE Similar to Tallqvist method Technical modifications done to improve accuracy and reliability Rapid, simple, inexpensive and reliable. Consists of printed set of colours with hemoglobin values between 4-14 g/dl. Developed by WHO after extensive field trials. USES Detection and management of anemia in underdeveloped countries. Screening blood donors Screening children and females in health programs Monitoring iron treatment Decision making about referral Point of care device

20. CYANMETHHEMOGLOBIN METHOD METHOD OF CHOICE PRINCIPLE: EQUIPMENT: SAHLI’S PIPETTE PHOTOELECTRIC COLORIMETER OR SPECTRO PHOTOMETER 5 ML PIPETTE REAGENTS: DRABKIN’S SOLUTION CYANMETHB STANDARD SOLUTION WITH KNOWN HB VALUE SPECIMEN: EDTA ANTICOAGULATED VENOUS BLOOD BLOOD OBTAINED FROM SKIN PUNCTURE TEST TUBE

21. PROCEDURE Diluted cyanmet standards are available for preparation of a calibration graph. Standard cyanmetHb solution can also be serially diluted with Drabkin’s solution.

22. COMPOSITION OF DRABKIN’S SOLUTION pH= 7.0-7.4 Potassium ferricyanide : 200 mg Potassium cyanide: 50 mg Potassium dihydrogen phosphate: 140 mg Non ionic detergent: 1 ml Distilled water: 1000 ml POINTS TO NOTE: Erroneous results can be seen in: Lipemic blood (HYPERTRIGLYCERIDEMIA) Leucocytosis (>25,000/ μ L) Plasma protein abnormality (MULTIPLE MYELOMA, WALDENSTORM’S MACROGLOBULINEMIA) 2. CyanmetHb is stable. So delay in taking the reading of absorbance will not affect the results. 3. Measures all forms except sulfHb

23. ADVANTAGES DISADVANTAGES All forms of Hb except sulfHb are converted to cyanmetHb No visual error as color matching is not required Stable cyanmetHb Absorbance may be measured soon after dilution. Reliable and stable reference standard is available from WHO for direct comparison. Diluted blood has to stand for a period of time Potassium cyanide is poisonous so it can NEVER BY PIPETTED BY MOUTH. Abnormal plasma proteins or leukocytosis can cause turbidity when dilute with Drabkin’s solution.

24. SPECIFIC GRAVITY METHOD: SG of CuSO4=1.053, equivalent to Hb level=12.5 g/dl Rapid and simple Gives approximate Hb value Used for selection of donors in blood banks. Abnormally high values can be seen in: Leucocytosis ( eg : CML) Hypergammaglobulinemia ( eg : Multiple myeloma) OXYMETHB METHOD: Rapid and simple No stable standard solution available Color of oxyHb fades quickly No Hb derivatives other than oxyHb are measured.

25. AUTOMATED ANALYSER Modified cyanmetHb method Other chemicals are sodium lauryl sulphate , imidazole, sodium dodecyl sulphate Measurements are made at various wavelengths depending on the final stable product. 3 PART DIFFERENTIAL ANALYSERS 5 PART DIFFERENTIAL ANALYSERS Two chambers: Hb /WBC chamber RBC/Platelet chamber - It is called so because it categorizes white blood cells into three main types: neutrophils, lymphocytes, and monocytes. Provides accurate red cell parameters, platelet parameters and reticulocyte parameters. Called so because it categorises white blood cells into five types: neutrophils, lymphocytes, eosinophils , monocytes and basophils.

26. TO READ Anemia Defintion Classification Causes Clinical features

27. STAINING OF PERIPHERAL BLOOD SMEAR

28. PERIPHERAL BLOOD SMEAR Specimen for microscopic examination prepared by spreading a drop of blood across a glass slide followed by staining using Romanowsky’s stains. Uses: Cause of anemia or thrombocytopenia Identifying type of leukemia Diagnosing hemoparasitic infections Monitoring effect of chemotherapy on bone marrow To provide a direction for further investigations that will help in arriving at the correct diagnosis.

29. PREPARATION OF A BLOOD SMEAR Length of slide: 75x25 mm Thickness of slide: 1 mm Slide should be clean, dry and grease free. Blood sample: EDTA anticoagulated venous blood or capillary (finger prick) blood. BEST IF DIRECTLY MADE FROM SKIN PUNCTURE Length of smear: 3 cm

30. IDEAL BLOOD SMEAR Tongue shaped with a smooth tail Does not cover the entire area of the slide Has both thick and thin areas with gradual transition Does not have any lines or holes.

31. STAINING OF BLOOD SMEAR Blood smears are routinely stained by one of the Romanowsky stains. Stains included are: May- Grunwald - Giemsa stain Jenner Wright’s Leishman’s Field’s COMPONENTS OF ROMANOWSKY STAINS ACIDIC BASIC Negatively charged AFFINITY FOR BASIC COMPONENT. Binds to cationic sites Gives orange red color to Hb and eosinophilic granules. Eg : Eosin Y Positively charged AFFINITY FOR ACIDIC COMPONENT. Binds to anionic sites Gives blue gray color to nucleic acids, nucleoproteins and basophilic granules. Eg : Methylene blue, Azure B

32. LEISHMAN STAIN Compound dye- eosin and methylene blue dissolved in acetone free methyl alcohol. EOSIN METHYLENE BLUE ACETONE FREE METHYL ALCOHOL Acidic dye Stains basic substances Gives orange red color to Hb and eosinophilic granules. Basic dye Stains acidic granules, leukocyte nuclei, basophil granules and neutrophilic granules (weakly) Methyl alcohol is a fixative Alcohol precipitates plasma proteins which acts as a glue and fixes the blood cells to the slide Alcohol also preserves the morphology and chemical status of the cells. Acetone is a strong lipid solvent so it will cause crenation, shrinkage and even lysis of cells. Hence, ACETONE FREE SOLUTION. Water also causes hemolysis and rouleaux formation. So, FREE FROM WATER also.

33. PROCEDURE

34. QUESTIONS! Why do we wait for 2 minutes after applying Leishman’s stain? Sometimes, a greenish scum can be seen over the smear. Is it bad? Why is buffered water used and not distilled water? Can we use tap water?

35. WHY DO WE WAIT FOR 2 MINS? Alcohol will fix the blood cells on to the glass slide. Alcohol will preserve the shape and chemistry of the cells to its living state as much as possible. SOMETIMES, A GREENISH SCUM CAN BE SEEN FLOATING OVER THE SMEAR. IS IT BAD? No! It indicates that staining has been done properly. Indicates ripening of the stain. WHY IS BUFFERED WATER USED AND NOT DISTILLED WATER? Buffered water is phosphate buffer where pH is adjust to 6.8 This pH allows stain to penetrate better. CAN WE USE TAP WATER? No. Cells will not be stained properly because of the unknown pH and salt content. Impurities in tap water can also show up as artefacts on the blood film.

36. FEATURES OF A WELL STAINED BLOOD SMEAR UNDER NAKED EYE UNDER LOW POWER (100X) UNDER HIGH POWER (400X) Smear appears translucent and bluish pink when seen against a white surface Thickness is uniform throughout. Red cells appear as dots, uniformly spread out in a single layer. WBC cannot be differentiated. No stain precipitate is present on the smear. Red cells are stained dull orange pink and show central pallor WBCs lie scattered among the red blood cells. Neutrophils: Pale pink cytoplasm and mauve purple granules Eosinophils : Pale pink cytoplasm and orange red granules Basophils: Blue cytoplasm and dark blue-violet granules. Monocytes: Gray-blue cytoplasm, fine reddish granules Small lymphocytes: Dark blue cytoplasm Platelets: purple

37. UNDERSTAINED Causes: Insufficient staining time Prolonged buffering or washing Old stain OVERSTAINED Causes: Thick blood smear Prolonged staining Insufficient washing Alkaline pH of stain components WELL STAINED

38. TO READ Abnormalities in shape and size of RBC. Leucocyte classification Causes of increase or decrease in each type of leucocyte Function of leucocytes Causes of increase or decrease in platelet count.