Tests of Regeneration

Macrocytic Hypochomic RBC

Which laboratory test best indicates whether
the bone marrow erythropoiesis is adequate
for the degree of anemia?
Reticulocyte count?
Harold Tvedten
MCV?
Strömsholm Referral
Animal Hospital
Graphic displays of RBC?
MCH?

Vote: Tell Your Neighbor Young Erythroid Cells

Which laboratory test best indicates whether the What stages of young erythroid cells can we
bone marrow erythropoiesis is adequate for the recognize in the blood of our patients?
degree of anemia? Metarubricytes
Reticulocyte count? Reticulocytes: Polychromatophils
MCV? Erythrocytes
Graphic displays of RBC? More stages?
MCH?
Other?

Case Presentation Hematology Results

Test Patient Reference
A Wachtelhund bitch had pale mucous HCT 32 % 39-59
membranes and a previous test showed it had
low hemoglobin, hematocrit and RBC count. WBC 16.5 x 109/L 6-17
8816365
Platelets 368 x 109/L 170-490
MCV 103 fl 63-75
MCHC 264 g/L 335-365

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Advia Advia

Summary of RBC graphics

graphics from from Advia
Advia 2120 2120 show a
regenerative
response. Is it
as expected?

Advia Advia How many RBC were

macrocytic and hypochromic?

How many
RBC are
macrocytic and
hypochromic
based on the
cytogram?
How can one
be more
specific?

Advia There were 76.6 + 4.8 % RBC which were Advia

macrocytic and 15.7 % hypochromic. Only
If there was
2.6 % were normocytic normochromic.
76.6 + 4.8 % +
15.7 % =
97 %
macrocytic
hypochromic
RBC, how
many
reticulocytes
are expected?

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 Vote: Tell Your Neighbor Advia
There were
How many reticulocytes are expected if 97% 28.6 %,
of RBC are macrocytic hypochromic? 897 x 109/L
5% reticulocytes.
15 % Are macrocytic
25 % hypochromic
45% RBC
75 % reticulocytes?
95 % Vice versa?

Advia Interpretation?
Why are there
97 % How many reticulocytes are expected in a mild
macrocytic anemia with hematocrit of 32 %?
hypochromic Why were there 97 % macrocytic hypochromic
RBC but only RBC but only 29 % reticulocytes?
28.6 %, How regenerative was this anemia?
reticulocytes? What type anemia is expected in this dog?

Late Stage Regenerative Anemia Late Stage Regenerative Anemia

This case had 28.6 % reticulocytes and
97 % macrocytic hypochromic RBC.
This is interpreted to be that the difference is
many cells that were reticulocytes now were
macrocytic hypochromic RBC that had lost their
ribosomes.
This case supports that concept.
Phosphofructokinase deficiency has hemolytic
events and even under calmer days the RBC have
a shorter half life. Up to 90 % reticulocytes have
been seen in dogs with pfk deficiency. Thus it is
possible for them to have 97 % macrocytic
hypochromic RBC weeks after a hemolytic
episode.

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 Lose Ribosomes

The Advia system, which shows macrocytic

hypochromic RBC, is excellent in showning that
a regenerative response occured more than a
week earlier. Reticulocytes live only 1-3 days in
circulation depending on how early they are
released from the bone marrow. Erythrocytes in
dogs live 100-115 days.

Peak Reticulocytes 4-7 Days Red Cell Aging in Vivo

Reticulocyte numbers peak at 4-7 days and then
decrease in numbers after a regenerative
response. Reticulocytes are macrocytic
hypochromic cells. I believe that these young
RBC are still macrocytic hypochromic after they
lose their ribosomes.
How long are young RBC macrocytic and or
hypochromic?
What really happens with canine RBC with time?
Canine reticulocytes live 1-3 days if they are
similar to human reticulocytes.
How long do RBC remain
Macrocytic? … Hypochromic?

Red Cell Aging in Vivo Red Cell Aging in Vivo

Identification of the kinetics of reticulocyte A third method is to use

changes is relatively easy because they contain transfusions and induced
RNA (ribosomes) and are easily identified. polycythemia to inhibit
Studing erythrocyte maturation/aging is not as bone marrow erythro-
easy. Using the theory more mature cells are poiesis to minimize
denser is one method of identifing more mature production of new RBC
cell. Marking RBC with biotin at the begining of so that transfused
their life span is another. reticulocytes can be
monitored.

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 Young Ganzoni showed retics from
Rat Reticulocyte Aging in Vivo anemic rats lived about 2 days
Reticulocytes
Ganzoni (Brit J Haemat 1969, 16:119-135)
hypertransfused rats to prevent erythropoiesis and
bled other rats 3 times taking 1/3 the blood
volume to obtain reticulocyte rich blood with
young reticulocytes to transfuse into they
polycythemic rats.

Macrocytic Macrocytic Ganzoni showed macrocytic

Hypochromic Hypochromic hypochromic cells survived up
to about 10 days.

Ganzoni showed MCV

macrocytic
hypochromic
cells survived up
to about 10 days.

2 8 10

Reticulocyte
Reticulocyte Aging in Vitro Aging in Vitro

Koury (Blood 2005 105 :2166-2174) isolated

mouse reticulocytes from in vitro cultures of
rubriblasts (human literature: proerythroblasts).
Most ribosomes and RNA were degraded within
20 hours (1 day) but only 20-25 % of ”cells
isolated as reticulocytes” became biconcave
erythrocytes by 110 hours (4.6 days).

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 Reticulocyte Aging in Vitro Reticulocyte
Aging
Koury in vitro study Reticulocytes lost
supports the concept most ribosomes by 20
that young RBC lose hours, but were losing
their ribosomes and cellular components
RNA days before by exocytosis at 40
they take the shape hrs and still had
(and size?) of organelle components
mature erythrocytes. by 110 hrs.

Young Erythroid Cells One More Category

What stages of young erythroid cells can we
recognize in the blood of our patients?
Repeating the question from the beginning.
Metarubricytes (orthochromic normoblasts)
Reticulocytes: Polychromatophils
Erythrocytes
More stages?
These stages of young erythroid cells we can
recognize in the blood of our patients!
Metarubricytes
Reticulocytes: Polychromatophils
Macrocytic Hypochromic RBC
Erythrocytes

Conclusion Rat Red Cell Aging in Vivo

Use Macrocytic Inbred rats were transfused

Hypochromic cells for first to Hct = 65-75% and
identification of late stage then retransfused to keep
regeneration in dogs. HCT > 50 %. This
prevented much production
of new RBC.
Have a little more on changes in more Ganzoni J Clin Invest
mature RBC with age, if time allows. 1971, 50:1373

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 Rat Red Cell Rat Red Cell
Rat erythrocytes Aging in Vivo Aging in Vivo
decrease in volume and
lose hemoglobin content
Theoretical Plot
with time.
Hemoglobin
concentration increases Loss of Water
early after a return to
Reticulocytes Splenic removal
near normal levels. of RBC membrane
Ganzoni J Clin Invest
1971, 50:1373

Rat Red Cell Oldest RBC

are not most Red Cell Aging in Vivo
Aging in Vivo dense

Based on Ganzoni’s rat RBC study

erythroid cells are macrocytic up to 7-8 days
of age and hypochromic up to 13-14 days
of age.

8 14

Red Cell Aging in Vivo Classification Systems

Veterinary Human
Ganzoni’s rat model was also used for Rubriblast Pronormoblasts: Proerythroblasts
macrocytes stimulated by anemia. He showed that Prorubricyte Basophilic normoblasts
rat macrocytes lose water and even hemoglobin Rubricyte Polychromatophilic normoblasts
while in circulation but remain viable. Metarubricyte Orthochromic normoblasts
Reticulocyte Reticulocyte
Rat RBC live about 60 days while canine RBC Erythrocyte Erythrocyte: RBC
live about 100 days. NRBC Normoblasts

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Two Years
90 % Retics
Ago
I presented a 4 It had 90 %
year old reticulocytes,
Wachtelhund 1,650 x 109/L
bitch at an and > 500 x
ESVCP meeting 109/L is a great
earlier with PFK increase.
deficiency.

PFK Deficiency Excessive Reticulocytosis

Analysis of 2 Wachtelhunds’ blood showed a
specific deficiency of phosphofructokinase
(PFK of 0.3 akat/erc compared to 2.6 akat/erc in
a normal control dog, performed by Gunnar
Ronquist). The Wachtelhound have a PFK
deficiency that is a different genetic mutation
than English springer spaniels and American
cocker spaniels.
One key to diagnosis in the earlier case was the
excessive reticulocytosis compared to other
hemolytic anemias. There were 90 % reticulocytes
at first presentation and then 810 x 109/L
reticulocytes when the hematocrit was 31 % (only
mild anemia). This reflects the very short half life of
the RBC (16 days instead of 20-28 days) in PFK
deficiency.

PFK Def PFK Def

PKK deficient PKK deficient

RBC have low RBC begin to lyse
2,3 DPG and with even mild
increased oxygen alkylosis seen
affinity which with
causes more hyperventilation.
tissue hypoxia Figures from
and thus ↑ Giger’s chapter in
erythropoiesis. Schalm’s.

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Clinical Signs PFK Defic.

The dog, 2 years ago, had a history of intravascular Case illustrated

hemolysis (red urine after a hunt) which was very PFK deficiency
typical for PFK deficiency. The blood picture also in a new breed
suggested PFK. It was fortunate we had tolerant and excessive
owners that permited a lot of testing over years to reticulocytosis
get the diagnosis. compared to
most dogs.