Hemoglobin estimation.

1. What is the normal Hb concentration Adults Males-13 to 18g/dl
      Adults Females-12 to 16 g/dl
      Children-12-14 g/dl
      Infants-16 to 22g/dl
2. Name the various methods for Hb estimation
  •  Acid hematin(sahlis method)
  • Cyanmethemoglobin method (calorimetric)
  • Oxyhemoglobin method
  • Alkali hematin method
  • Halden  carboxy haemoglobin method
  • Gasometric method
  • Specific gravity method
  • Tallquist method
  • Auto analyzers
3. Which is the best method for Hb estimation
  • Cyanmethemoglobin method
4. Which anticoagulant should be used for Hb estimation
  • Ethylenediamine tetra acetic acid(EDTA)
5. How much quantity of EDTA Should be Used for 1 ml of blood
  1. 1.0- 1.2 mg/ml of blood
6. How N/10 HCL is prepared
  • Concentrated HCl-4.5 ml
  • Distilled Water-500ml
  • To make 500 ml of N/10 HCL
7. What difference would it make if N/10 HCL is taken above or below the 20% mark?
  • If N/10 HCL more – The colour of undiluted solution may be lighter than standard.
  • If N/10 HCL less –All haemoglobin will not get converted.
8. What is the principle of sahlis method
  • Hemoglobin is converted by HCl into acid haematin which gives brown colur to solution
  • This colour of the solution is matched with the colour of the comparator of sahlis haemoglobinometer
9. What is the procedure of Acid hematin method
  • N/10 HCl is taken up to 20 mark of the gratuated tube
  • About 20 cc of blood is taken in the hemoglobin pipette
  • The tip of pipette is wiped with blotting paper
  • The blood is added to the HCl in the Hemoglobinometre  tube
  •  Wait for 10 minutes and then add distilled water drop by drop till the colour matches with the colour of  comparator of sahlis haemoglobinometer
  •  Haemoglobin is expressed in gm%
10. In sahlis method ,why do we wait for 10 minutes after mixing blood with acid
  • RBCs are lysed and Hb gets converted in to acid haematin completely
11. In sahlis Hb meter apparatus, why are square tube and flat comparator glasses preferred?
  • It is easier to compare adjoining flat surfaces ex: Square tube with acid haematin with flat brown surface on either side. Error due to curvature is avoided
12. Which precaution should be taken during the procedure
  • Wait for atleast 10 min for formation of Acid hematin
  • Tap water cannot be used as the pH varies and accurate values may not be obtained
  • Remove stirrer while adding distilled water and also while taking reading
  • Hemoglobinometer should be hold at the eye level against good light and lower meniscus of the Hemoglobinometer tube solution should be considered.
13. Why N/10 HCL used in Sahlis method
  • Brown colour of the comparator glass is equivalent to the colour of acid haematin produced using  N/10 Hcl for blood sample of 14.8 gm%. Test solution is compared against it. Therefore no other strength of HCL Should be used
14. What are the advantages of Sahlis method
  • Simple bed side test
  • Reagents and apparatus are cheap
  • Easy to perform
  • Quick and inexpensive
15. What are the disadvantages of Sahlis method
  • Time factor
    • The color of acid hematin fades quickly with passage of time
    • Color of the standard comparator fades with passage of time
    • Acid hematin solution is not stable
  • Color Matching: Variation from person to person in matching the color
  • Technical errors:
    • Improper mixing of blood
    • Tissue fluid containing capillary blood
    • Errors in pipette calibration ,sample and equipment,
  • Cannot estimate all different types of haemoglobin(carboxy, meth,and supha)
  • This method is not suitable for fetal haemoglobin which is not converted in to acid hematin
  • Non hemoglobin substances like protein and lipids in the plasma influence the colour of the blood that is diluted with acid
16. How much blood is used in cyanmet Hb estimation method
  • 20 microliter
17. Which reagent is used in cyanmet Hb method?
  • Drabkin solution
18. Drabkin solution composition?
  • Drabkin’s solution PH-7.0-7.4
  • Potassium cyanide – 50mg
  • Potassium ferricyanide – 200mg
  • Potassium dihydrogen phosphate – 200mg
  • Distilled water 1L,
  • Non ionic detergent  1ml.
The final solution should be clear and pale yellow in colour.
19. What is the principle of cyan met Hb method
  • First Hb is converted to methemoglobin by potassium ferricyanide and then KCN converts it to cyanmethemoglobin
20. Advantages of cyanmethemoglobin method
  • All forms of Hb except sulphemoglobin are converted cyanmethemoglobin (HiCN)
  • Visual error is not there as no colour matching is required
  • Cyanmethaemoglobin solution is stable and its colour does not fade with time so readings may not be taken immediately
  • A reliable and  reference standard is available from world health organisation for direct comparison
21. In cell counters what is the chemical used(cyanide free) for estimation of hemoglobin
  • Sodium Lauryl sulphate is used and it rapidly converts Hb into detectable chromogen
22. Define anemia
  • It is a Pathological condition characterized by decrease in oxygen carrying capacity of blood manifested by  decreased Hb concentration, reduced RBC count and packed cell volume
23. How will you classify anemia
  •   On the basis of morphology
    • Normocytic normochromic
    • Microcytic hypochromic
    • Macrocytic hypochromic / normochromic
  • According to underlying mechanism
    • Anemia due to blood loss
    • Anemias due to increased rate of destruction
    • Anemias  due to impaired red cell production
24. What are the indications of haemoglobin estimation?
  • To determine presence and severity of anaemia
  • Screening for polycythemia
  • To assess response to specific therapy in anaemia
  • Estimation of red cell indices
  • Selection of blood donars
25. Conditions where Hb is decreased
  • Anemia
  • Auto immune diseases
  • water retention(pregnancy, edema)
  • Blood loss(bleeding,internal hemorrhage)
  • Parasitic infection
  • Drugs
  • Lead poisoning
  • Dietary deficiency(iron,cu,vitamins)
  • Malabsorption of nutrients
  • Chronic disease
26. Conditions where Hb is increased
  • High altitude
  • Severe vomiting  or diarrhoea(hemoconcentration)
  • Infants
  • Smoking
  • Polycythaemia vera
  • Obstructive lung disease
  • Congestive heart disease
  • Splenic hypo function
27. Name some tumors which cause rise in hemoglobin values
  •  Leiomyoma uterus
  • Renal cell carcinoma
  • Liver tumors(Hepato cellular carcinoma)
  • Cerebellar hemangioblastoma
28. What are the abnormal haemoglobins
  • Hb S,Hb C,Hb D.Hb E
29. What are conditions that come under hemoglobinopathies
  • Sickle cell anemia,Thalassemia
References
  1. S.Sanyal. Pathology Practical book, 4th edition
  2. Harsh Mohan. Practical pathology, Third edition with viva voice questions
  3. Dr .Tejindar Singh,  Dr .K. Uma  Chaturvedi.Practical pathology for undergraduate and post graduate students-First edition
  4. Ganga S. pilli. Viva in pathology, First edition
  5. Internet sources
 
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