Hi Elena

I'll try and follow up on this for you but there's a new BCSH guideline published recently: Guidelines for the diagnosis and treatment of cobalamin and

folate disorders. British Journal of Haematology, 2014, 166, 496–513. I attach the pdf for you.

Megaloblastic Anaemia usually is not hyperchromic because the increased hemoglobin content in RBCs (Increased MCH) is according to increased cells volume: MCHC is within the normal range. Hyperchromia (increased MCHC) occours, i.e. on the hereditary spheroctosis because the surface of the membrane (consequentially, MCV) is reduced.

D'Onofrio G, Zini G. Morfologia delle Malattie del sangue. Verduci Ed. 2013 Roma

There is simply not such a thing as hyperchromic anemia! Microcytic anemias can be hypochromic or normochromic; normocytic and macrocytic anemias including megaloblastic anemias are normochromic.   

Yes, Jacques!  The same concept can be expressed with different words and we are completely in agreement :

Thanks to all for your useful comments.

Hypochromic Macrocytic

I agree with Dr Elion completely, I personally have never used the term (hyperchromic) in my practice and always instruct my students to define anemia as either normochromic or hypochromic.

We need to consider two different criteria. The first one is pathophysiological and excludes the concept of hyperchromia. The second one is practical and, I think, clinically usefull: an isolated increase of  MCHC can be usefull to suspect an hereditary spherocytosis ( Conway et Al. The clinical relevance of an isolated increase in the number of circulating hyperchromic cells. J Clin Pathol 2002, 55: 841-844). In addition the word "Hyperchromic" is conventionally used in microscopy to describe the RBCs  lacking central area not colorful.

I can agree with Antonio. A word “hyperchromic” is usually in use by lab specialists to describe a microscopy finding: lack of a central pallor in erythrocytes. Some people use a word “hyperchromasia” instead of hyperchromia", but it may be confusing because of its specific connotation of “hyperchromasia” with pathology staining of a nuclei! Suggesting malignancy.

For patients with anemia, the peripheral smear morphology provides key information to create the differential diagnosis.

To confirm the CBC findings.

To review relevant white blood cell (WBC) and platelet (PLT) findings.

To review RBC morphology:

1. Shape. What is/are the dominant poikilocyte(s)?

2. Size. Is there anisocytosis or a dual population?

3. Color. Is there hypo- or hyperchromia? Is there anisochromia or polychromasia?

4. Inclusions. Are there Howell–Jolly bodies, malaria parasites, nucleated RBCs, etc.?

5. Arrangement. Is there agglutination or rouleaux?

Concluding: hyperchromia is not pathognomonic for B12 deficiency anemia but it can be observed in blood film examination in cobalamin deficiency subjects.

I am agree with Ewa and Antonio

But in Medicine, nothing is perfect or exact. Almost a third of B12 deficiency anemia could be normocytic because there is an iron deficiency associated. In those patients we can found a slight trend to hypochromia. Of course, the smear is very usefull to identify the presence of hypersegmentated, ovalocytes, anysocitosis and anisochromia

For measuring Mean corpuscular hemoglobin concentration (MCHC) the hemoglobin is divided by the hematocrit and multiplied by 100. As both the Hb and hematocrit decreases together, the net result remains the same; that means normochromic. 

 I agree with José Antonio García-Erce

The RBCs in megaloblastic anaemia are usually normochromic and many a time we see hypochromic macro-ovalocytes in these conditions but never hyperchromasia, 

It should be hyperchromic. Though errythroblast cytoplasm is matured, Hb Accumulation may continue till  nucleus maturaion become completed and itescapes from the bone marrow.

Megaloblastic anemia is characterised by normochromic macroovalocytes as the MCHC of the RBCs remains normal.In megaloblastic anemia with decrese in Hb,the HCt also decreases and so the MCHC=Hb/Hct remains normal.

Many contributions, many opinions and many definitions have been added to this discussion.

Often the same concept was repeated with different words. I try to summarize:

1. The "chromia" of red blood cells is not their content in Hemoglobin (MCH) but the percentage of cell volume occupied by Hemoglobin (MCHC);

2. The maximum volume of a red blood cell that can be occupied by Hemoglobin is about 1/3 of the total volume. This because geometric rules (surface/volume ratio);

3. Hemoglobin may occupy the volume normally available (about 33 percent = 33 of MCHC = normochromia) or a lower volume (

Thanks for this academic and smart explanation!

It is normochromic type of anaemia as the Hb synthesis is not effected. If Megaloblastic anaemia was due to nutritional deficiency, hypochromia due to co-existing iron deficiency is commonly seen. Whereas, in pernicious anaemia due to isolated B12 deficiency, the PB smear shows normochromic macrocytes. No hyerchromasia in B12 deficiency.

Dr. Ewa Stepien has previously well defined difference from "hyperchromasia" and hyperchromia (his answer 15 days ago). Hyperchromasia if the feature of nuclear increased staining and is predominantly used in histological/cytological descriptions. To describe RBCs chromia the usual terminology is: normo-hypo and hypercrhomia with the meanings described earlier. Polychromasia is also used to describe different dyeing affinity of RBCs population on the peripheral blood smears. Accordingly, all morphologist know that in megalocytic anaemia we can observe many RBCs without central pallor. The morphologists conventionally define these red blood cells "hyperchromic RBC". This, only morphologic term, NOT DEFINE RBC WITH INCREASED HEMOBLOBIN CONTENT!

Dear it presents as normocytic hyperchro ia

Pure b12 deficiency anemia is associated with normochromic erythrocytes

It is Macrocytic (Macroovalocytic) Normochromic.

If polychromasia appeared in the film due to reticulocytosis ??

Polychromasia may be increased on the peripheral blood smear even when the reticulocyte count or percentage is normal. This finding suggests the premature release of erythroid cells from a marrow that is not hyperactive

Please Gamal, can you explain your statement better?

I'm reasonably sure that an increasing of reticulocytes (and therefore erythrocytes with polychromasia) is always associated with accelerated erythropoiesis. This physiological adjustment occurs only when there is loss (acute or chronic) of red blood cells or even when the availability of oxygen decreases as in the stay at high altitude. Another cause is the administration of erythropoietin for the purpose of doping. In all these situations, the increasing of reticulocytes and, therefore, polychromasia is the result of an appropriate bone marrow response to an appropriate stimulus

Another issue deserves to be discussed: usual meaning of "normal range/or count" should not be applied to the reticulocytes counting which can be only "adequate" or "not adequate". So, 30 or 50x109 reticulocytes are adequate for maintaining the "normal" values of hemoglobin (and therefore the number of RBCs) in a non-anemic patient. On the contrary, the same values are not sufficient (they are not adequate) to restore normal hemoglobin values in case of severe anemia that has, for example, hemoglobin equal to 50-60g/L. If these concepts are not clear you run the risk of the diagnostic error of not correctly classifying an anemia as hypo-regenerative (just as in the example). Similarly, in that same anemia, a reticulocyte count equal to 250x109 can be considered "adequate" for the restoration of normal hemoglobin values.

Dear Antonio La Gioia

As we know the reticulocytosis is increased due to , RBC hemolysis, hemorrhage or response to treatment including iron, folate, and vitamin B12 .

The most common supravital stain is performed on reticulocytes using new methylene blue or brilliant cresyl blue, which makes it possible to see the reticulofilamentous pattern of ribosomes characteristically precipitated in these live immature red blood cells by the supravital stains. But the RBC with reticulofilamentous morphology by leishman stain appear polychromatic.

Summary:

A. By methylene blue or brilliant cresyl blue we see reticulocytes

B. By Leishman stain: The cells appeared as Polychromasia

Let's try to get some clarity. Reticulocytes are red blood cells recently passed from bone marrow to peripheral blood. They still retain a residue of material ribosomal that appears as granules and/or filaments with methylene blue or brilliant cresyl blue staining. This material is abundant immediately after the passage in the PB and gradually decreases until it disappears within 24 hours. Heilmeyer, based on the content of the reticulocytes, classified them from class I to IV, i.e., from the youngest with abundant material (class I) to the older ones with poor and pointy material (class IV). See the attached photo, taken from my old experience before the automated count advent. As stated by Gamal, the panoptic stains (Giemsa, Wright, Leysman, and others) are not suitable for staining the ribosomes inside the reticulocytes, which, however, take on a different shade of color than mature RBCs (polychromasia). Finally, it should be pointed out that only I-II-class reticulocytes appear polychromatophilic with panoptic staining, while those of classes III-IV have no different coloration from mature RBCs. In short, all polychromatophilic RBCs are reticulocytes, while not all reticulocytes appear as polychromatophilic RBCs in the panoptic staining.

Thank you Dr Antonio La Gioia .for interesting explaination and nice picture.

Thank you Dr Gamal Abdul Hamid. Your discussions on RG are always inspiring and allow interesting exchanges of opinions.

Let me give some unpleasant radical view of mine. Historically the moephological discription of two dimensional microscopic picture of red cells using two rough criteria: (1) diameter when compared with nucleus of small lymphocyte and (2) Ratio of diameter of the central halo of red cell to the diameter of the red cell. As one can easily understand they were arbitrary with lot of inter-observer variation. It was made a bit more precise with Wingtrobe's red cell indices (MCV, MCH and MCHC) in 1929. Again the precision was poor in RBC count due to high dilution and fatigue in accurately counting large number of red cells needed to be counted. While estimation of Hb and PCV by Wintrobe's method were found to have reasonably good precision. Hence MCV and MCH done manually were not very dependable, while it was beleived that MVHC is very good. Now we know the problem created by trapped plasma in the PCV method, which increases in most of the anemias due to anisocytosis. So the "depedable" index MCHC was falsely lower than the actual one. This very well matched with the deceptive larger halo of very often seen leptocytes when discribed the cell by microscopic view. In fact here the ratio of cell mebrane area : volume is increased. So the terminologies of eithter "Normochromic" or "Hypochromic" were cotinued due to two false ways agreeing with each other. Suprisingly the term "Hyperchromic" was not allowed to be used even in case of hereditary sphercytic anemia !

After the advent of electronic cell counters (around 1956), we knew the true PCV and MCHC, as there was not rtapped plasma there. So if you are using cell counters, the morphological classification of anemia (the first stgep of the algorithm towards knowing the cause of anemia, which is not a single disease, but manifestation of large number of causes) is very simple: only 3 groups: Normocytic, Macrocytic and Microcytic. The "Chromia" part is now does not exist and MCV and MCH are more useful than MCHC. One can use anisocytosis in terms of RDW as suggested by Beesman, with one precaution that it should not be in terms of %CV (!), but SD. that will subtype each of three types in "Homogenous" and "Heterogenous"

Although the theoretical assumptions well described by Ramesh in his valuable asswer seem to support well the classification of anemia proposed by Bessman, the methodological uncertainties about the use of RDW still remain. RDW-SD is the fL value of the amplitude of the RBC histogram measured at a height of 20% of the total height of that histogram. Although this measure appears to be capable of reducing the inter-instrumental analytical variability of the RDW parameter, the methodological differences of the different hematological analyzers remain large and poorly comparable.

Antonio is perfectly right. Now it is right time that this technological problem is addressed by institutions like ICSH, so that we can use it in a meaningful way. One can not deny that if this is successfully done, it will be a very sensitive index of early identification of anisocytosis. MCHC is still useful to detect need for re-calibration of cell counter, if we look at the L-J graph of daily average.

I fully agree with Ramesh on the need for hematological parameters to be standardized. Unfortunately, as far as erythrocytes are concerned, the methodological differences between different analyzers seem currently insurmountable. Siemens and Abbott systems for RBC counting use the optical method after isovolumetric sphericalization. Mindray and Sysmex systems that use the impedentiometric method associated with hydrodynamic focusing have significant differences with other impedentiometric analyzers such as Beckman Coulter systems. These are the reasons why some RBC parameters such as MCV and RDW are currently poorly comparable or not comparable between different analyzers

normochromic