Dr. med. Benedikt Lohr: INSTAND EQA Scheme Expert, MVZ for Laboratory Medicine and Microbiology Koblenz-Mittelrhein, Koblenz
The determination of the CBC is one of the most common laboratory medical examinations. The Hb concentration included in the CBC is the product of the erythrocyte count and the Hb content of the erythrocytes. The hemoglobin content per unit of blood volume is measured, so the result depends on the plasma volume.
There are countless invasive and non-invasive methods for determining the Hb concentration, both historical and current, and those under development. Review articles such as Karakochuk et al. contain a non-exhaustive overview.
In the context of the Instand e.V. EQA Schemes 162, 209, 211 and 612/912, the methods briefly described below are particularly worth mentioning:
HiCN method (Cyanhaemiglobin Method)
A defined sample volume is mixed with a defined volume of conversion solution: after hemolysis of the erythrocytes, potassium ferricyanide and potassium cyanide oxidize hemoglobin via hemoglobin (synonym: methemoglobin) to form stable cyanhaemiglobin (HiCN), the absorbance of which is measured photometrically at 540 nm. The method detects almost all Hb variants (except sulphaemoglobin, a rarely occurring dyshaemoglobin). The HiCN method is the reference method. Due to the use of toxic cyanide, among other things, it is usually replaced by cyanide-free alternatives in routine practice.
SLS (Sodium Lauryl Sulphate) Detection Method
In this example of a cyanide-free method, sodium lauryl sulfate first lyses the erythrocytes. After a conformational change in the hemoglobin molecule, oxidation of the divalent iron, and thus the formation of methemoglobin, SLS binds the resulting trivalent iron and forms a stable SLS-Hb color complex with maximum absorption at 555 nm. The absorption level is proportional to the hemoglobin concentration of the sample.
QBC - Quantitative Buffy Coat
In this method, EDTA blood is centrifuged in fluorescence-coated capillaries, whereby cell layers are separated by density. The Hb concentration is calculated via the immersion depth of a float. The method is fast, comparatively simple, requires manageable equipment, and is therefore suitable for field use on ships or military bases. (There are also numerous publications on using the QBC method for diagnosing blood parasites, particularly malaria).
Spectrophotometry in BGA Analyzers
Blood gas analyzers use oximetry, which measures Hb over multiple wavelengths in the visible spectrum and thus distinguishes between different Hb derivatives. After chemical or mechanical lysis of the erythrocytes to avoid scattering processes, the spectrophotometric measurement is carried out, the physical basis of which is the Lambert-Beer law. There is no chemical conversion of the hemoglobin or its derivatives. The concentration of total hemoglobin (tHb) is the sum of oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb), and the dyshaemoglobins carboxyhemoglobin (COHb), methemoglobin (MetHb), and sulphaemoglobin (SulfHb), whereby most oximeters do not report the latter.
from Zijlstra and Buursma. 1997. spectrophotometry of hemoglobin: absorption spectra of oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin and methemoglobin from cattle. Comp. Biochem. Physiol.
The performance characteristics of the methods and the comparability between the different methods are the subject of many primary scientific studies and various review articles, e.g., in Whitehead et al.
In Germany, the guideline of the German Medical Association for the quality assurance of laboratory medical examinations (Rili-BÄK) is the authoritative document for quality management and quality assurance of laboratory medical examinations.
Haemoglobin is part of Table B 1-2 a of Part B 1 on quantitative tests. This specifies not only the frequency of participation in the EQA scheme but also, in particular, the permissible relative deviation and the target value type for the EQA scheme.
For hemoglobin, a participant's result may not deviate by more than 6% from the result of the reference method value to receive a certificate from the reference institution for this measurement. This means that the various, quite heterogeneous methods of haemoglobin determination in the proficiency test must be measured against the cyanhaemiglobin method, see above.
Another complicating factor is that the materials sent in the EQA scheme differ significantly in composition. For example, the EQA samples for RV 162 are aqueous solutions based on bovine hemoglobin, while stabilized control blood and fresh, EDTA-anticoagulated blood are sent for RV 211 and RV 612/912, respectively. Thus, interesting and important aspects such as the comparability of human and bovine hemoglobin, the changes caused by the stabilizer contained, and the time-critical pre-analysis when sending fresh blood by post are affected.
The cross-sectional guidelines on therapy with blood components and plasma derivatives of the German Medical Association not only specify the transfusion thresholds in g/dl or mmol/l for the various adult and pediatric patient groups for acute and chronic anemia but also explicitly mention the extent and rate of blood loss as one of many additional criteria for determining the indication for transfusion.
Thus, efforts to ensure good precision and accuracy of any method used are not academic but directly relevant to patients.
The hemoglobin concentration must be comparable between the value of the ABG analyzer in the intensive care unit and the determination in the central laboratory on any hematology analyzer. If the alleged blood loss is due to the lack of comparability of the measurement results, this would run counter to high-quality medicine, which underlines the need for internal and external quality assurance to achieve the best possible quality of results and treatment.
Literature:
Karakochuk et al. doi.org/10.1111/nyas.14003
Whitehead et al. doi: 10.1111/nyas.14124
Zijlstra and Buursma. 1997. Spectrophotometry of Hemoglobin: Absorption Spectra of Bovine Oxyhemoglobin, Deoxyhemoglobin, Carboxyhemoglobin, and Methemoglobin. Comp. Biochem. Physiol.