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   Northern Medical Journal, 2006; Vol-15 (2)      

 

           Very High Leucocytes count Interfere with Colorimetric

                     Measurement of Hemoglobin Level

                                                Dr. Md. Mujibur Rahman

Abstract:

Among the colorimetric methods of hemoglobin estimation Cyanmethemoglobin method is commonly used. A new colorimetric method using acid-hematin is also having been found to be similar sensitivity. During estimation of hemoglobin, red cells are lysed releasing their hemoglobin that gives color after combination with chemical used in both methods for measuring hemoglobin. Leucocytes are not excluded during measurement of absorbance, so the optical density conferred by the leucocytes should interfere with actual level of hemoglobin in blood. Though in normal range of population blood leucocytes do not confer any significant optical density during reading absorbance but very high leucocytes count (e.g., leukemia) interfere actual hemoglobin level due to showing significant optical density. In this study, done for a period of over five years, hemoglobin levels were estimated from 14400 patients out of whom 118 blood samples were of leukemias and 4 were leukemoid reactions those showed leucocytes count between 50×109/L to 600×109/L. On average leucocytes count 50×109/L of blood found to increase hemoglobin level to 0.55gm% of blood above actual level.

 

Introduction:

The hemoglobin content of a solution may be estimated by several methods; by measurement of its colour, by its power of combining with oxygen or carbon monoxide and by its iron content. Ideally as a functional estimation of Hb, measurement of oxygen capacity is most accurate and better than the method by measuring iron content of the Hb; but these two methods are impracticable for routine clinical purposes. For clinical purposes Sahli's acid-haematin method and Cyanmeth-hemoglobin methods are commonly applied. Cyanmeth-hemoglobin method is a photoelectric method and more accurate than Sahli's acid-haematin method. A new photoelectric method developed by the author is also being used and found to be as accurate as Cyanmeth-hemoglobin method. In both the methods hemoglobin can be measured by colorimeter (530 nm) and as well as by auto-analyzer (547nm).  In both methods, colour developed by hemoglobin by combination with their respective chemicals used is measured. The leucocytes and platelets that are present in blood are not found to be lysed and can confer significant optical density during measurement of absorbance. Usually within normal limit the absorbance for leucocytes and platelets is ignored but high leucocytes count particularly in patients of leukemia, has been found to show significant optical density during measurement of hemoglobin. Platelets are tiny particles and if their absorbance is ignored, optical density conferred by very high leucocytes should not be ignored because of falsely increased level of hemoglobin.

 Actually, this study was initiated after an incidental finding of normal hemoglobin level of a leukemia patient with very high WBC count but clinically he was very anemic. During hemoglobin estimation the prepared fluid was found to be slight hazy than of patient of normal WBC count. Then Hb% was estimated by measuring absorbance after centrifuging the fluid and ultimately the result correlated well with clinical condition. The study was carried out to estimate actual level of hemoglobin of blood with very high leucocytes count in patient of leukemia or leukemoid reaction.

 

 

 

 

Materials and Method:

The study was carried out in a private laboratory during the period from 1998 to 2005 in Rangpur, Bangladesh. Hemoglobin was measured from patients attending the laboratory in different times and days. More than 14500 blood samples were taken out of which 42% were female and 58% were male. Out of these, 112 cases were of leukemia and 3 cases were of leukemoid reaction with leucocytes count between count 50,000 to 6, 00,000/ cumm of blood. Hemoglobin was estimated in both Cyanmeth-hemoglobin and new photoelectric method using ERMA Colorimeter F-15. It should be noted that no separate standard was used in new colorimetric method but Cyanmeth standard hemoglobin has been used in this new method. Absorbance was measured two times - first, five minutes after preparation of the final reagent with adequate mixing and another is taken using supernatant fluid after centrifugation   for 5 minutes at 3000 rpm. Total leucocytes counts were measured simultaneously by Neuber-Chamber and differential count were made after Leishman's staining.

 

Results:

Obtained hemoglobin levels were plotted before and after centrifugation and variations were compared with their total leucocytes count. In cases of leukemia and leukemoid reactions the types of cells were noted. Out of 14500 cases 12430 cases showed leucocytes count up to 11,000/cumm. Maximum variation Hb% of these cases before and after centrifugation was less than 0.1gm%. Cases showing leucocytes count from 11,000 to 20,000/cumm were 1850. Maximum variation after centrifugation was 0.2gm%. Cases showing leucocytes count from 20,000 to 30,000/cumm were 24. Maximum variation after centrifugation was 0.3gm%. Cases showing leucocytes count from 30,000 to 40,000/cumm showed maximum variation after centrifugation was 0.45gm%. Cases showing leucocytes count from 50,000 to 1, 00,000/cumm showed maximum variation after centrifugation was 1.2gm%. Cases showing leucocytes count from 1, 00,000 to

 2, 00,000/cumm showed maximum variation after centrifugation was 2.5gm%. Cases showing leucocytes count from 2, 00,000 to 3, 00,000/cumm showed maximum variation after centrifugation was 3.5gm%. Cases showing leucocytes count from 3, 00,000 to 4, 00,000/cumm showed maximum variation after centrifugation was 4.5gm%. Cases showing leucocytes count from 4, 00,000 to 5, 00,000/cumm showed maximum variation after centrifugation was 5.8gm%. Cases showing leucocytes count from5, 00,000 to

6, 00,000/cumm showed maximum variation after centrifugation was 6.8gm%.

Discussions:

Within the normal range of value the estimation of accurate level of hemoglobin of a person is usually not mandatory for clinical purposes. But in most medical or surgical cases estimation of actual hemoglobin level is very useful for the clinicians. Particularly in cases of leukemia, haemorrhagic disorders or many other cases where the physicians need to observe the effect of blood transfusion, effect of treatment with haematinics or effect anticancer drugs or many other disease conditions those are related significant variation of hemoglobin level.

From this study it is of no doubt that very high WBC count in blood shows falsely increased value of Hb% than actual Hb% level at the rate of 0.5% to 0.6% per 50,000 WBC/ cumm of blood. No differences were observed between the two methods of hemoglobin estimation. One can easily confirm my observation by estimating Hb% before and after centrifuging the fluid during colorimetric estimation of hemoglobin of a leukemic patient with high WBC count and again the result may be compared with a more accurate method like measurement of oxygen capacity or by measuring iron content of the Hb. For the last few decades it is not clear how hemoglobin of patients with high WBC count had been estimated or being estimated in the laboratories where colorimetric method is followed. Probably the discrepancies are being minimized by both the physician and laboratory practitioner on the clinical ground by observing the color of sclera in these cases. So far the need of accurate level of Hemoglobin is concerned; this observation should be brought to the knowledge of the laboratory practitioners as well as the physicians.

 

Table no I.

Total count of WBC/cumm of Blood

No of cases

Maximum variation of

Hb% in gram

Calculated maximum variation of HB%  for 50,000 WBC /cumm of Blood

Up to 11,000

12430

<0.1

0.5

11,000 to 20,000

1848

0.2

0.5

20,000 to 30,000

24

0.3

0.5

30,000 to 40,000

11

0.45

0.56

40,000 to 50,000

06

o.60

0.60

50,000 to 1,00,000

10

1.2

0.60

1,00,000 to2,00,000

18

2.5

0.62

2,00,000 to 3,00,000

17

3.6

0.62

3,00,000 to 4,00,000

19

4.7

0.56

4,00,000 to 5,00,000

09

5.8

0.52

5,00,000 to 6,00,000

08

6.8

0.56

 

Average Hb% variation after centrifugation 0.558 gm% per 50,000 WBC /cumm

 

 

 

Table No II.

 

Variation Hb% according to type of WBC in cases of Leukemia

and leukemoid reaction.

Type of Cells

Calculated maximum variation of Hb% in gram for 50,000 WBC/cumm of Blood

Myeloid series    (72)

0.51

Lymphoid series (50)

0.62

Average variation per 50,000 WBC count 0.556 gm%

 

 

 

 

 

References:

  1. Mujibur Rahman. A New Method for Measuring Hemoglobin.
  2.  BarbaraJ, Brain & Imelda Bates. Basic haematologic techniques. Dacie and Lewis Practical Haematology. Ninth Edition, Churchill Livingstone. 2001.19-46.
  3. Firkin Frank et al.de Gruchy's Clinical Haematology I Medical Practice.The Red Cell and Anaemia. Fifth Edition.Blackwell Science Ltd. 1996. 17-36.

 

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