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Species specific White Blood Cells (WBC) composition

Species specific White Blood Cells (WBC) composition


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In our ongoing immunology undergrad course I learnt that neutrophil primarily fights off bacterial infection and lymphocyte is produced in response to viral infection. I also learnt that neutrophil count in humans is about 70 % in human WBCs while in horse lymphocytes account for 68% of WBC. It is supposed to make humans more immune to bacterial infection and similarly horses better adapted to fight off viral infections. However I cannot find out any relevant material as to what could be the reason that such biased WBC cell component composition has evolved in different species. Please help me find out relevant material on this.


Five Types of White Blood Cells

White blood cells (WBCs) serve as the principal actors of the immune system. The 5 classes of WBCs, or leukocytes, differ in appearance and function. These classes include neutrophils, monocytes, lymphocytes, eosinophils and basophils. WBCs function primarily to protect and defend the body against infectious invaders, including bacteria, viruses, fungi and parasites. A reduced number of white blood cells in the body, called leukopenia, can leave the body vulnerable to wide variety of infectious diseases.

If you are experiencing serious medical symptoms, seek emergency treatment immediately.


Introduction

To thwart pathogens and keep infections at bay hosts rely on a competent immune system 1 . While the relationship between immune function and individual survival has been well documented 2,3,4 , there has been relatively little research focused on sex differences in immune defence in free-living animals.

Differences in immune response between the sexes have been described extensively across vertebrates. These sex differences have been traditionally associated with the immunomodulating effect of sex hormones, where oestrogens, found in higher concentrations in females, act as weak immune-enhancers, and androgens, higher in males, as immune-suppressors 5,6 . However, these studies have been centred primarily on humans and laboratory animals, while there is increasing evidence suggesting that the association between sex hormones and sex differences in immunity in the wild are not as simple as first thought. Two independent meta-analysis showed that testosterone did not have a consistent overall immunosuppressive effect in males, and the effect depended on the taxa studied and whether the experimental manipulations involved hormone concentrations above physiological levels 7,8 . A recent study has also challenged the notion of sex biases in immunity by finding no overall sex difference in immune estimates in a large-scale comparative analysis including vertebrates and invertebrates 9 . However, Kelly et al. 9 showed that some patterns do arise when focusing on specific immune variables and taxonomic groups, such as mammals, which showed a strong male bias in specific pro-inflammatory cytokines. Kelly et al. 9 did not find overall sexual differences in birds immunity, but they concluded that future studies of sex differences in immunity should include variables known to affect immune functioning, such as age 10 , nutritional state 11 , photoperiod 12 or seasonality 13 . The latter variable is especially relevant, because seasonal changes, in particular the transition between the non-breeding and the breeding period, involve major physiological and behavioural changes. They may also include pronounced environmental shifts, particularly in species that migrate between breeding and non-breeding grounds, which is the case in many species of birds. Accordingly, several studies have found important sex-specific changes in immunity between the non-breeding and breeding period in birds. For example, Hõrak et al. 14 found that female Great Tits, Parus major, had more circulating lymphocytes than males in spring but not in summer. Merrill et al. 15 found that male Brown-headed Cowbirds, Molothrus ater, showed higher bactericidal capacity than females during the breeding period compared to the non-breeding period. Reasons behind such complex seasonal, species-specific and sex-specific immunity are not fully understood. Recurring explanations include sex-specific energetic and nutritional costs that may be traded off against immunity 16,17,18 , thus resulting in an impaired immunity in the sex with higher energy expenditure (e.g. courtship displays, egg production, parental care 19,20,21 ).

Alternatively, immune defence may be compromised in situations that cause strain or tension, i.e. stress 22 . Corticosterone, the main circulating glucocorticoid in birds, could play an important role here. First, because corticosterone is involved in regulating the metabolism 23 , and second, as result of an increase in stress-induced corticosterone production (e.g. during territory defence) that could supress immune function 24,25,26 . However, a comprehensive analysis that simultaneously investigates seasonally-related and sex-specific immunity across bird species is largely lacking. Also, it is unknown whether potential sex-specific or seasonal patterns are consistent between immune parameters 27 .

Here, in order to better understand the variation in avian immune function, we conducted a meta-analysis to test for seasonal (breeding versus non-breeding season) and sexual differences in immunity across bird species. Because of the known effects of ontogeny and captivity on immunity 28,29 , we restricted our analysis to data from free-living adult birds. We included information from nine measurements characterising immune status: the relative frequency of four types of white blood cells (heterophils, lymphocytes, macrophages, eosinophils), the ratio of heterophils/lymphocytes (H/L ratio, a glucocorticoid-mediated immune index of stress), and four widely used immune response indexes (the phytohaemagglutinin test, bacteria-killing ability assay, haemolysis assay, and the haemagglutination assay). For each of these nine immune parameters we estimated their overall meta-analytic means (i.e. estimates of sex-specific immune biases). Based on previous studies 9,30 , we expected no sex difference in white blood cells levels and a small female bias in the immune response indexes. Next, we broke down these overall estimates by season, and computed one estimate for the non-breeding period and one for the breeding period. This allowed us to test if these seasonal estimates were sex-biased, and if season, as a variable, had a significant effect on the immune parameters. Because breeding often incurs increased workload and higher energy demands compared to non-breeding birds in winter 16 , we expected the two periods to differ from each other, and season to significantly affect immune variables 31,32 .

Furthermore, we used the estimates from male and female individuals to test if the sexes could respond differently to the transition between seasons. Males are generally more involved in courting behaviour and intrasexual aggression therefore, we predicted a possible stress-mediated immunosuppression 26 in males that could outweigh an alternative immunosuppression due to energetic trade-offs in females 21 . Thus, in the transition from non-breeding to breeding, males may exhibit stronger changes in immune estimates than females.


Results

Clinical parameters tested are indicative of physiological response during exposure to HH

A detailed overview of clinical parameters at baseline (BL), 24 h and 72 h is shown in Table 1. After direct ascent to high altitude, peripheral oxygen saturation (SpO2) showed a significant modification over time (ANOVA P < 0.001) with a reduction at 24 h (−11%, P < 0.001) and a progressive increase between 24 h and 72 h (+5%, P = 0.001). SpO2 did not return to the baseline values for any participant during stay at 3800 m. The heart rate (HR) increased from baseline values up until 24 h (+35%, P < 0.001), then HR decreased (−10%, P < 0.24), remaining higher compared to baseline (+21%, P < 0.006). Similarly, the breath rate (BR) enhanced within 24 h and after 72 h compared to baseline values (respectively +22%, P = 0.01 and +19%, P = 0.02). All the subjects included in data analysis had a value of Lake Louise Score (LLS) < 3 during three days of exposure to high-altitude hypoxia.

Exposure to HH results in differential expression of key TFs in human WBCs that is also time exposition related

Gene expression of different TFs (HIF-1α, HIF-2α, NRF2 and p65 subunit of NF-κB) were determined at baseline (262 m) and after 24 h and 72 h of high altitude exposure (3830 m). HIF-1α mRNA levels presented a parabolic relationship with time of exposure (72 h) to HH (ANOVA P = 0.001). Specifically, HIF-1α mRNA levels increased significantly after 24 h (+59%, P = 0.01) of hypoxic exposure, and subsequently returned to values comparable to baseline levels after 72 h (Supplementary Table S1 and Fig. 1A). HIF-2α mRNA levels consistently increased over time (ANOVA P = 0.001), reaching a peak after 72 h of exposure (+41%, P < 0.001) (Supplementary Table S1 and Fig. 1B). Similarly, the NRF2 mRNA level increased constantly during hypoxic exposure (ANOVA P = 0.04), reaching a peak after 72 h (+87%, P < 0.001) (Supplementary Table S1 and Fig. 1C). Finally, in comparison to baseline values, p65 mRNA levels were not significantly different after either 24 h or 72 h of high altitude exposure (ANOVA P = 0.71) (Supplementary Table S1 and Fig. 1D).

The relative quantification of mRNA levels for Transcription Factors HIF-1α, HIF-2α, NRF2 and NF-κB (p65) in white blood cells reveal that a hypobaric hypoxia stimulus inducts different TF expression patterns that is time dependent. (A) HIF-1α shows a transcription peak within 24 h, while HIF-2α (B) and NRF2 (C) show an expression peak after 72 h after the beginning of the stimulus. On the contrary, the NF-κB gene doesn’t show differences in its expression during the study (D). RQ was calculated as fold change using the 2 −(ΔΔCt) method. The results of mRNA are the average of the values assessed after three reaction tests. Values are mean ± SD. *P < 0.05 and **P < 0.001 after Bonferroni correction.

Direct measurement of circulating pro-inflammatory cytokines during prolonged HH exposure indicate swift mediation of inflammatory state in HA responders

Gene expression analysis of pro-inflammatory markers (IL-1β and IL-6) were determined at baseline (262 m) and after 24 h and 72 h of high altitude exposure (3830 m). IL-1β and IL-6 mRNA levels were not significantly modified during the 72 h period (ANOVA P = 0.65 and ANOVA P = 0.60 respectively) in WBCs (Supplementary Table S1 and Fig. 2A,B). On the contrary, IL-1β plasma levels significantly changed over time (ANOVA P = 0.001). Specifically, IL-1β plasma levels increased after 24 h (+25%, P < 0.02), and returned to baseline after 72 h (−2%, P = 0.77) (Supplementary Table S2 and Fig. 2C). IL-6 plasma levels did not significantly changed over time (ANOVA P = 0.07) (Supplementary Table S2 and Fig. 2D). However, we observed a significant reduction of IL-6 at 72 h of high altitude exposure, in comparison to values reported after 24 h (−5%, P = 0.04) (Supplementary Table S2 and Fig. 2D). Both plasma levels of IL-1β and IL-6 were considered in the normal range for the general population throughout the entire study period 41,42 . Within 24 h of exposure, 2 participants received a single dose of Paracetamol (500 mg). The adjustment of results for the use of paracetamol was performed, and no significant modifications were obtained (data not shown).

Relative quantification of mRNA levels and protein plasma levels for pro-inflammatory cytokines in white blood cells reveal a slight increase in inflammatory state during HH exposure. (A,B) IL-1β and IL-6 mRNA quantification don’t reveal significant variation in mRNA production for both cytokines during hypoxia stimulus over time. The results of mRNA are the average of the values assessed after three reaction tests. Relative quantification of mRNA expression for pro-inflammatory cytokines was calculated as fold change using the 2 −(ΔΔCt) method. Values are mean ± SD. (C,D) Only IL-1β circulating protein showed a slight transient increase after 24 h of HH exposure. The absolute values for both circulating proteins are in the normal range for general population. The median (continuous band) and the mean (dotted band) are represented inside the boxes. *P < 0.05 after Bonferroni correction.

Direct 24 h exposure to HH induces marked increases in ROS, TBARS, 8-isoPGFα, 8-OHdG, PC and concomitant reduction in TAC

To determine biomarkers of oxidative stress at baseline and during high altitude exposure, we measured the levels of ROS production, TBARS (Thiobarbituric acid reactive substances, markers of lipid peroxidation), 8-isoprostanes (8-isoPGF2α, lipid oxidation products and potential disease mediators), 8-hydroxy-2′ -deoxyguanosine (8-OHdG, products of DNA oxidation), PC concentrations (protein oxidative carbonylation) and TAC (total antioxidant capacity, the sum of aqueous and lipid soluble low-molecular weight antioxidants) for the whole cohort (Supplementary Table S3). OxS biomarkers levels at baseline (262 m) were in agreement with previous values estimated for a healthy population 43 . The basal level values for 8-isoPGF2α and 8-OHdG considered for statistical analysis were obtained from a matching group of the general population.

Sub-acute HH exposure (24 h), led to changes in indices of OxS namely: increased levels of ROS production as detected by EPR, TBARS, 8-isoPGF2α, 8-OHdG, PC, plus a decrease in total antioxidant capacity (TAC). Changes were detectable and reached a peak of +218% (ROS), +70% (TBARS), +54% (8-isoPGF2α) and +61% (PC) after 24 h (P < 0.001, P < 0.001, P = 0.001, P < 0.001 respectively) compared to baseline (Supplementary Table S3 and Fig. 3). Levels of 8-OHdG marker of DNA damages peaked after 24 h (+178%) the change was only slightly significant (P = 0.024) compared to BL. TAC values showed an exactly opposite pattern over time (ANOVA P < 0.001) with the lowest level of −60% after 24 h (P < 0.001) (Supplementary Table S3 and Fig. 3) and no restoring of the basal levels of antioxidant activity after the 72 h period of exposure (−25%, P = 0.001). Additionally, ROS production rate, TBARS, 8-isoPGF2α, 8-OHdG and PC concentrations significantly changed over time of exposure to HH within 72 h (ANOVA P < 0.001, P < 0.001, P = 0.002, P = 0.011, and P < 0.001 respectively) (Supplementary Table S3 and Fig. 3).

Box and Whisker plots show the marked effects of hypobaric hypoxia exposure (3830 m) on ROS production and relative biomarkers of oxidative damage. Direct 24 h exposure to HH induces marked increases in ROS, TBARS, 8-isoPGF α, 8-OHdG, PC and concomitant reduction in TAC. After 72 h of exposure to hypoxia there is a partial recover of the basal conditions with a slight improve of the TAC and a reduction of ROS and cellular damages amounts. The median (continuous band) and the mean (dotted band) are represented inside the boxes. *P < 0.05 and **P < 0.001 after Bonferroni correction.

Correlation among TFs gene expression, inflammatory and oxidative markers and clinical variables

We chose to perform a correlation analysis in order to investigate the tendency to show a coordinated expression among TFs, other studied genes (i.e. IL-1β and IL-6) related to OxS and inflammatory circulating markers (i.e. IL-1β and IL-6), over time. In particular, the correlation analysis was chosen to provide information pertaining to which biological processes were interconnected and which displayed interdependence. The main aim was to identify which potential interactions may be of mechanistic importance in the early phase of human response to hypobaric hypoxic exposure.

Clinical variables showed a strong correlation with ROS and TAC but not with TFs and inflammation markers

SpO2 showed strong correlations with both ROS production (ANOVA r = −0.76, P < 0.001) and TAC (ANOVA r = +0.82, P < 0.001) over time (Table 2). TAC and ROS were also correlated (ANOVA r = −0.66, P < 0.001) (Table 2 and Supplementary Fig. S1).

SpO2 showed a negative but marginally significant correlation with three out of four TFs investigated (HIF-1α, NF-κB and NRF2) (ANOVA r = −0.41, P = 0.03 ANOVA r = −0.47, P = 0.01 and ANOVA r = −0.42, P = 0.03 respectively).

SpO2 showed a marginally significant correlation with IL-1β and IL-6 protein levels in plasma (ANOVA r = −0.46, P = 0.02 ANOVA r = −0.44, P = 0.02). IL-1β and IL-6 protein concentrations showed a marginal significant correlation with ROS (ANOVA r = +0.47, P = 0.02 and r = +0.34, P = 0.09 respectively) and TAC (ANOVA r = −0.35, P = 0.08 and r = −0.47, P = 0.02 respectively) over time. Only a marginal significant correlation was identified between IL-1β mRNA and IL-6 mRNA (ANOVA r = +0.40, P = 0.04) levels in WBCs. On the contrary no correlations were observed between the mRNA of the two investigated interleukins and their protein concentrations in plasma (ANOVA r = +0.32, P = 0.12 and ANOVA r = −0.04, P = 0.84 respectively). Finally, IL-6 protein showed a marginally negative correlation with TAC (ANOVA r = −0.47, P = 0.02).

Transcription factors showed a differential correlation with each other, OxS markers and inflammatory variables

HIF-1α mRNA was not correlated with HIF-2α (ANOVA r = −0.19, P = 0.34) and NRF2 mRNA (ANOVA r = −0.29, P = 0.15) whereas HIF-2α mRNA levels were positively correlated with NRF2 gene expression over time (ANOVA r = +0.58, P = 0.002) (Table 2 and Supplementary Fig. S1).

HIF1α mRNA was positively correlated with ROS overtime (ANOVA r = +0.62, P < 0.001) (Table 2). HIF-2α mRNA levels showed a marginally significant correlation with the IL-6 mRNA levels over time (ANOVA r = +0.46, P = 0.02) (Table 2 and Supplementary Fig. S1).

NRF2 mRNA level was negatively correlated with TAC (ANOVA r = −0.43, P = 0.03). Significant correlations were identified involving NF-κB mRNA with TAC and ROS (ANOVA r = −0.59, P = 0.002 and ANOVA r = +0.46, P = 0.02). Only marginal positive correlations were identified involving NF-κB mRNA and plasma interleukins IL-1β and IL-6 overtime (ANOVA r = +0.49, P = 0.01 and ANOVA r = +0.47, P = 0.02 respectively).


New HIV Model Shows Virus Doesn't Kill White Blood Cells, It Just Homes Them To Death

ANN ARBOR---University of Michigan scientist Denise Kirschner has developed a new mathematical model that shows how HIV---the virus that causes AIDS---slowly destroys its victim's immune system by accelerating a normal process called homing, which diverts white blood cells from the bloodstream to the lymph system.

Increased understanding of the complex relationship between the HIV virus and the immune system is important, because it will help scientists develop more effective treatments for AIDS and suggest new targets for therapeutic drugs.

"This model indicates that the key to extending survival time for people with AIDS is to minimize the number of CD4 cells exposed to signals in the lymph system which lead to apoptosis or cell suicide," says Miles W. Cloyd, Ph.D., a professor of microbiology at the University of Texas Medical Branch at Galveston.

Developed in collaboration with G.F. Webb, Ph.D., of Vanderbilt University, Kirschner's model validates the homing theory of HIV progression, which was first proposed by Cloyd and his colleagues. Results from the model were published in the August 1 issue of The Journal of AIDS.

Many scientists believe HIV destroys the immune system by attacking white blood cells called CD4 or helper T-cells in the bloodstream. But Kirschner and Cloyd maintain that HIV's lethal action is much more subtle and indirect.

Their model shows that CD4 cells actually self-destruct in the lymph system. Death comes as a result of exposure to biochemical signals involved in the homing process, which trigger apoptosis or cell suicide.

"Previous HIV models have focused on what happens in the bloodstream, but the real action is in the lymph system," says Kirschner, Ph.D., an assistant professor of microbiology and immunology in the U-M Medical School. "A very small percentage of cells dies from apoptosis on a daily basis, but over a seven-year period, it adds up to almost 100 percent."

Results from the U-M model are consistent with what happens in people, according to Cloyd. Data from clinical studies with HIV-infected patients show that the population of uninfected CD4 cells in their blood falls to 15 percent of normal during a seven-year period.

When the HIV virus binds to a CD4 cell, one of three things can happen, according to Kirschner. First, the cell can be actively infected and turn into a cellular factory that produces more virus. Second, the immune cell can be latently infected the virus gets inside the cell nucleus, but remains dormant. Third, and most common, the CD4 cell can be abortively infected. In this case, the virus enters the cell cytoplasm, but doesn't enter the nucleus.

"When the HIV virus binds to a CD4 cell, the process activates a receptor molecule called L-selectin on the cell membrane, which signals the CD4 cell to home to the lymph system," Kirschner explains. "If we could block that signal, we could preserve healthy CD4 cells."

In future research, Kirschner plans to model the role of co-receptors involved in HIV binding to CD4 cells. The virulence of infection varies depending on the co-receptor chosen by the virus. She also plans to explore the immune response to HIV, and how different types of immune responses, known as TH1 or TH2 responses, determine disease progression.

"Miles Cloyd's work has brought the concept of lymphocyte circulation, which was a hot topic in the 1970s, back into the scientific limelight," she says. "There could be applications to many other diseases."

Development of the mathematical model of HIV progression was funded by the National Heart, Lung and Blood Institute of the National Institutes of Health, the National Science Foundation and the American Foundation for AIDS Research.

Story Source:

Materials provided by University Of Michigan. Note: Content may be edited for style and length.


Species specific White Blood Cells (WBC) composition - Biology

Hi all.
We have never done a manual WBC count before and my advisor has asked me to
get a count of WBCs (primarily nuetrophils and lymphocytes) in wright's
stained peripheral blood smears. What is the general protocol for a manual
count? Do you count WBC's inb relation to RBCs or just WBCs, the whole
slide, or just representative sections?

P.S - this is for rat sample, not human

Is what your supervisor wants a WBC Differential? If so you count 100
cells identifying each different type of cell and report the percentage
of each cell type. A white blood cell count would have to be performed
on a whole blood specimen - not a peripheral smear - there is no way to
measure volume otherwise as cell counts are reported as number of cells
per volume measured.

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Missy
Sent: Tuesday, September 26, 2006 10:25 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] WBC counts

Hi all.
We have never done a manual WBC count before and my advisor has asked me
to
get a count of WBCs (primarily nuetrophils and lymphocytes) in wright's
stained peripheral blood smears. What is the general protocol for a
manual
count? Do you count WBC's inb relation to RBCs or just WBCs, the whole
slide, or just representative sections?

P.S - this is for rat sample, not human

Is this some kind of project for a class? If not, hand it over to
hematology. If so, get a medical technology text book and look up blood
cell counting. To do it right requires special counting slides that are
etched with grids.

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Missy
Sent: Tuesday, September 26, 2006 7:25 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] WBC counts

Hi all.
We have never done a manual WBC count before and my advisor has asked me
to get a count of WBCs (primarily nuetrophils and lymphocytes) in
wright's stained peripheral blood smears. What is the general protocol
for a manual count? Do you count WBC's inb relation to RBCs or just
WBCs, the whole slide, or just representative sections?

P.S - this is for rat sample, not human

It sounds like your advisor wants a "differential" WBC if you are attempting
to do it on a stained peripheral blood smear. To do this you would need
some type of multi-key manual differential counter (example:
http://stores.implex.net/minnesotamedical/product_info.php?cPath=205_174_176
&products_id=1726)

To do this you assign a key on the counter to a specific white cell (i.e.
nuetrophils) and do the same for the rest of the keys with the different
types of white cells. You then scan the slide and count the different white
cells (punching the proper key for each) until you reach a total count of
100 cells (pay no attention to the red cells). Your differential would then
be the number of specific cells counted express as a percentage. Example,
you counted 62 nuetrophils and 38 lymphocytes = 62% neturo & 38% Lymphs
respectively.

To do a total WBC, you would have to have a hemocytometer (what Tim was
talking about), calibrated hemo pipettes, a lysing reagent, normal saline
diluent, and WHOLE blood. You would dilute the whole blood to the proper
dilution with normal saline, add the lysing reagent to destroy the red
cells, load the hemocytometer, and count all the white cells you see.

Or, as Tom suggested. give the assignment to the Hematology Department.
-)

Ford M. Royer, MT(ASCP)
Histology Product Manager
Minnesota Medical, Inc.
7177 Madison Ave. W.
Golden Valley, MN 55427-3601
CELL: 612-839-1046
Phone: 763-542-8725
Fax: 763-546-4830
eMail: ***@bitstream.net

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Morken, Tim
Sent: Tuesday, September 26, 2006 11:21 AM
To: Missy ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

Is this some kind of project for a class? If not, hand it over to
hematology. If so, get a medical technology text book and look up blood
cell counting. To do it right requires special counting slides that are
etched with grids.

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Missy
Sent: Tuesday, September 26, 2006 7:25 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] WBC counts

Hi all.
We have never done a manual WBC count before and my advisor has asked me
to get a count of WBCs (primarily nuetrophils and lymphocytes) in
wright's stained peripheral blood smears. What is the general protocol
for a manual count? Do you count WBC's inb relation to RBCs or just
WBCs, the whole slide, or just representative sections?

P.S - this is for rat sample, not human

Good luck finding a hemacytometer and/or any calibrated pipettes. Ours
ended up in the display of "antique instruments and equipment". That's
the best place for them (speaking as an old dinosaur who actually
learned how to use them).

Jacquie Poteete MT(ASCP)QIHC
Lead Medical Technologist, IHC Laboratory
Saint Francis Hospital, Tulsa, OK

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Ford
Royer
Sent: Tuesday, September 26, 2006 2:24 PM
To: 'Missy' ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

It sounds like your advisor wants a "differential" WBC if you are
attempting to do it on a stained peripheral blood smear. To do this you
would need some type of multi-key manual differential counter (example:
http://stores.implex.net/minnesotamedical/product_info.php?cPath=205_174
_176
&products_id=1726)

To do this you assign a key on the counter to a specific white cell
(i.e.
nuetrophils) and do the same for the rest of the keys with the different
types of white cells. You then scan the slide and count the different
white cells (punching the proper key for each) until you reach a total
count of 100 cells (pay no attention to the red cells). Your
differential would then be the number of specific cells counted express
as a percentage. Example, you counted 62 nuetrophils and 38 lymphocytes
= 62% neturo & 38% Lymphs respectively.

To do a total WBC, you would have to have a hemocytometer (what Tim was
talking about), calibrated hemo pipettes, a lysing reagent, normal
saline diluent, and WHOLE blood. You would dilute the whole blood to
the proper dilution with normal saline, add the lysing reagent to
destroy the red cells, load the hemocytometer, and count all the white
cells you see.

Or, as Tom suggested. give the assignment to the Hematology
Department.
-)

Ford M. Royer, MT(ASCP)
Histology Product Manager
Minnesota Medical, Inc.
7177 Madison Ave. W.
Golden Valley, MN 55427-3601
CELL: 612-839-1046
Phone: 763-542-8725
Fax: 763-546-4830
eMail: ***@bitstream.net

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Morken,
Tim
Sent: Tuesday, September 26, 2006 11:21 AM
To: Missy ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

Is this some kind of project for a class? If not, hand it over to
hematology. If so, get a medical technology text book and look up blood
cell counting. To do it right requires special counting slides that are
etched with grids.

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Missy
Sent: Tuesday, September 26, 2006 7:25 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] WBC counts

Hi all.
We have never done a manual WBC count before and my advisor has asked me
to get a count of WBCs (primarily nuetrophils and lymphocytes) in
wright's stained peripheral blood smears. What is the general protocol
for a manual count? Do you count WBC's inb relation to RBCs or just
WBCs, the whole slide, or just representative sections?

P.S - this is for rat sample, not human
_______________________________________________
Histonet mailing list
***@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

We purchase hemocytometers from Fisher (Healthcare catalog) or VWR. We
still use them for lung lavage/washings from mice. AND we do cell count
aka differentials on Diff Quik (if you can get it without difficulty)
stained murine lung lavage/cytospins. If you have to do a differential on
animal blood smears, buffy coats, Wright Giemsa stain is superb for this
purpose, Harleco brand.

For a WBC count, contact a clinical laboratory to run the WBC counts on
their automated counters, much more accurate and worth the cost. It was
painful to work with the RBC and WBC pipettes - HOORAY for modern
technology and instrumentation.

Hi
I suspect that all your advisor wants to do is to get the relative
amounts of each of the leukocytes.
To do this select an area of the blood smear where the red blood cells
are separated a little. Avoid clumps where the smear will be thick or
the tail ends of the smear where the white cells may be clumped and
distorted.

Ideally the red blood cell should be salmon pink. If they are red then
all the colors that you have in a description of the white cells will be
shifted towards the red, if the red blood cells are slate blue then
similarly all the colors will be shifted towards the blue.

Start by counting the white cells in the first field.
Move the slide one field over say to the right. Count these cell types.
Then move one field down and count.
Then one field to the right and count.
Then up one field and count.
Repeat this procedure until you have counted at least 200 preferably
more leukocytes.
If you get to the end of the slide or in an undesirable area during
moving then move down one field and repeat this but moving to the left
etc.
This is the battlement method of counting and will give you a
differential count of leukocytes.
The more leukocytes you count the more accurate your percentages.
You may have trouble in finding basophils as they are only present in
the order of 0.5 to 1%.
Hope that this helps
Barry

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Poteete,
Jacquie A.
Sent: Tuesday, September 26, 2006 2:39 PM
To: Ford Royer Missy ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

Good luck finding a hemacytometer and/or any calibrated pipettes. Ours
ended up in the display of "antique instruments and equipment". That's
the best place for them (speaking as an old dinosaur who actually
learned how to use them).

Jacquie Poteete MT(ASCP)QIHC
Lead Medical Technologist, IHC Laboratory
Saint Francis Hospital, Tulsa, OK

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Ford
Royer
Sent: Tuesday, September 26, 2006 2:24 PM
To: 'Missy' ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

It sounds like your advisor wants a "differential" WBC if you are
attempting to do it on a stained peripheral blood smear. To do this you
would need some type of multi-key manual differential counter (example:
http://stores.implex.net/minnesotamedical/product_info.php?cPath=205_174
_176
&products_id=1726)

To do this you assign a key on the counter to a specific white cell
(i.e.
nuetrophils) and do the same for the rest of the keys with the different
types of white cells. You then scan the slide and count the different
white cells (punching the proper key for each) until you reach a total
count of 100 cells (pay no attention to the red cells). Your
differential would then be the number of specific cells counted express
as a percentage. Example, you counted 62 nuetrophils and 38 lymphocytes
= 62% neturo & 38% Lymphs respectively.

To do a total WBC, you would have to have a hemocytometer (what Tim was
talking about), calibrated hemo pipettes, a lysing reagent, normal
saline diluent, and WHOLE blood. You would dilute the whole blood to
the proper dilution with normal saline, add the lysing reagent to
destroy the red cells, load the hemocytometer, and count all the white
cells you see.

Or, as Tom suggested. give the assignment to the Hematology
Department.
-)

Ford M. Royer, MT(ASCP)
Histology Product Manager
Minnesota Medical, Inc.
7177 Madison Ave. W.
Golden Valley, MN 55427-3601
CELL: 612-839-1046
Phone: 763-542-8725
Fax: 763-546-4830
eMail: ***@bitstream.net

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Morken,
Tim
Sent: Tuesday, September 26, 2006 11:21 AM
To: Missy ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

Is this some kind of project for a class? If not, hand it over to
hematology. If so, get a medical technology text book and look up blood
cell counting. To do it right requires special counting slides that are
etched with grids.

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Missy
Sent: Tuesday, September 26, 2006 7:25 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] WBC counts

Hi all.
We have never done a manual WBC count before and my advisor has asked me
to get a count of WBCs (primarily nuetrophils and lymphocytes) in
wright's stained peripheral blood smears. What is the general protocol
for a manual count? Do you count WBC's inb relation to RBCs or just
WBCs, the whole slide, or just representative sections?

P.S - this is for rat sample, not human
_______________________________________________
Histonet mailing list
***@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

_______________________________________________
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http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Just a general tech-note. if you have the clinical lab run your whole
blood samples through their automated counters, make sure you inform them
what species the sample is from. You mentioned that you are working with
rats, so it should not be a problem with them getting an accurate WBC count.
In some species (i.e. birds) the RBCs are nucleated and will not completely
lyse with standard lysing reagents and therefore the RBCs could be
erroneously counted as WBCs giving a elevated count. Regardless, it is
important that your hematology department knows the species of the sample
that you are asking them to run through their analyzer.

Ford M. Royer, MT(ASCP)
Histology Product Manager
Minnesota Medical, Inc.
7177 Madison Ave. W.
Golden Valley, MN 55427-3601
CELL: 612-839-1046
Phone: 763-542-8725
Fax: 763-546-4830
eMail: ***@bitstream.net

-----Original Message-----
From: histonet-***@lists.utsouthwestern.edu
[mailto:histonet-***@lists.utsouthwestern.edu] On Behalf Of Gayle Callis
Sent: Tuesday, September 26, 2006 3:22 PM
To: Poteete, Jacquie A. ***@lists.utsouthwestern.edu
Subject: RE: [Histonet] WBC counts

We purchase hemocytometers from Fisher (Healthcare catalog) or VWR. We
still use them for lung lavage/washings from mice. AND we do cell count
aka differentials on Diff Quik (if you can get it without difficulty)
stained murine lung lavage/cytospins. If you have to do a differential on
animal blood smears, buffy coats, Wright Giemsa stain is superb for this
purpose, Harleco brand.

For a WBC count, contact a clinical laboratory to run the WBC counts on
their automated counters, much more accurate and worth the cost. It was
painful to work with the RBC and WBC pipettes - HOORAY for modern
technology and instrumentation.

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)


Conclusions

In this study, we proposed the development of a novel biological fuel cell that can utilize the body's own resources to generate electricity, through specific electrochemical interactions between cells and electrodes in close proximity. The motivation of this study is to develop a BFC that can be used to power implantable medical devices, including micro- and nano- biosensors for either therapeutic or physiological monitoring purposes. The study seeks to demonstrate that electron transfer between human white blood cells and an interfacing electrode can occur through any or all of three possible mechanisms: 1) direct electron transfer through membrane bound redox species (such as the flavocytochrome of NADPH oxidase) 2) indirect electron transfer through exocytosed non-metabolic biochemical species (e.g. serotonin) and 3) indirect electron transfer through exocytosed metabolically relevant biochemical species. Our results indicate that activated white blood cells can generate small electrical currents when introduced into the anode compartment of a proton exchange membrane fuel cell, with ferricyanide in the cathode compartment. Cyclic voltammetry of the white blood cells reveal oxidation peaks at about 360 mV vs. SCE. Peaks at this potential have been attributed to serotonin release. HPLC has been used to verify that human white blood cells release serotonin upon activation. Various white blood cell lines do not release serotonin, indicating that the isolated cells may uptake serotonin from the blood stream rather than metabolize it themselves.


Species specific White Blood Cells (WBC) composition - Biology

White blood cells, also called leukocytes, are cells that exist in the blood, the lymphatic system, and tissues and are an important part of the body's defense system. They help protect against infections and also have a role in inflammation, and allergic reactions. The white blood cell (WBC) count totals the number of white blood cells in a sample of your blood. It is one test among several that is included in a complete blood count (CBC), which is often used in the general evaluation of your health.

Blood is made up of three main types of cells suspended in fluid called plasma. In addition to WBCs, there are red blood cells and platelets. All of these cells are made in the bone marrow and are released into the blood to circulate.

There are five types of WBCs, and each has a different function:

  • Three types of WBCs are referred to as "granulocytes" because of the granules present in their cytoplasm. These granules release chemicals and other substances as part of the immune response. Granulocytes include:
      (neu) normally make up the largest number of circulating WBCs. They move into an area of damaged or infected tissue, where they engulf and destroy bacteria or sometimes fungi. (eos) respond to infections caused by parasites, play a role in allergic reactions (hypersensitivities), and control the extent of immune responses and inflammation. (baso) usually make up the fewest number of circulating WBCs and are thought to be involved in allergic reactions.
    • B lymphocytes (B cells) produce antibodies as part of the body’s natural defense (immune) responses.
    • T lymphocytes (T cells) recognize foreign substances and process them for removal.
    • Natural killer cells (NK cells) directly attack and kill abnormal cells such as cancer cells or those infected with a virus.

    When there is an infection or an inflammatory process somewhere in the body, the bone marrow produces more WBCs, releasing them into the blood, and through a complex process, they move to the site of infection or inflammation. As the condition resolves, the production of WBCs by the bone marrow subsides and the number of WBCs drops to normal levels again.

    In addition to infections and inflammation, there are a number of conditions that can affect the production of WBCs by the bone marrow or the survival of WBCs in the blood, such as cancer or an immune disorder, resulting in either increased or decreased numbers of WBCs in the blood. The WBC count, along with the other components of the CBC, alerts a healthcare practitioner to possible health issues. Results are often interpreted in conjunction with additional tests, such as a WBC differential and a blood smear review. A differential may provide information on which type of WBC may be low or high, and a blood smear may show the presence of abnormal and/or immature WBCs.

    If results indicate a problem, a wide variety of other tests can be performed to help determine the cause. A healthcare practitioner will typically consider an individual's signs and symptoms, medical history, and results of a physical examination to decide what other tests may be necessary. For example, as needed, a bone marrow biopsy will be performed to evaluate the bone marrow status.

    You may be able to find your test results on your laboratory's website or patient portal. However, you are currently at Lab Tests Online. You may have been directed here by your lab's website in order to provide you with background information about the test(s) you had performed. You will need to return to your lab's website or portal, or contact your healthcare practitioner in order to obtain your test results.

    Lab Tests Online is an award-winning patient education website offering information on laboratory tests. The content on the site, which has been reviewed by laboratory scientists and other medical professionals, provides general explanations of what results might mean for each test listed on the site, such as what a high or low value might suggest to your healthcare practitioner about your health or medical condition.

    The reference ranges for your tests can be found on your laboratory report. They are typically found to the right of your results.

    If you do not have your lab report, consult your healthcare provider or the laboratory that performed the test(s) to obtain the reference range.

    Laboratory test results are not meaningful by themselves. Their meaning comes from comparison to reference ranges. Reference ranges are the values expected for a healthy person. They are sometimes called "normal" values. By comparing your test results with reference values, you and your healthcare provider can see if any of your test results fall outside the range of expected values. Values that are outside expected ranges can provide clues to help identify possible conditions or diseases.

    While accuracy of laboratory testing has significantly evolved over the past few decades, some lab-to-lab variability can occur due to differences in testing equipment, chemical reagents, and techniques. This is a reason why so few reference ranges are provided on this site. It is important to know that you must use the range supplied by the laboratory that performed your test to evaluate whether your results are "within normal limits."

    For more information, please read the article Reference Ranges and What They Mean.

    The reference ranges 1 provided here represent a theoretical guideline that should not be used to interpret your test results. Some variation is likely between these numbers and the reference range reported by the lab that ran your test. Please consult your healthcare provider.

    Age Conventional Units 2 SI Units 3
    0-18 years Not available due to wide variability. See child's lab report for reference range.
    Adult 4,500-11,000 white blood cells per microliter (mcL) 4.5-11.0 x 10 9 per liter (L)

    1 from Wintrobe's Clinical Hematology. 12th ed. Greer J, Foerster J, Rodgers G, Paraskevas F, Glader B, Arber D, Means R, eds. Philadelphia, PA: Lippincott Williams & Wilkins: 2009.


    Supplementary information

    The authors thank Georg Hofer (Department of Anesthesiology and Critical Care Medicine, General Hospital of Silandro, Italy), Karla Balkenhol, Michael Pohl and Emily Procter (Eurac Research, Institute of Mountain Emergency Medicine, Bolzano, Italy), and Piergiorgio Lochner (Department of Neurology, University of the Saarland, Homburg, Germany) for invaluable support during data and sample collection and Tomas Dal Cappello (Eurac Research, Institute of Mountain Emergency Medicine, Bolzano, Italy) for support with the statistical analysis. The authors thank the Department of Innovation, Research and University of the Autonomous Province of Bozen/Bolzano for covering the Open Access publication costs.


    Species specific White Blood Cells (WBC) composition - Biology

    In the event of bacterial infection, large numbers of neutrophils migrate from the blood to the infected site in order to destroy the invading microorganism and thus protect the host. The neutrophils removed from the peripheral blood are then replaced by other neutrophils, at various stages of maturation, from the bone marrow pool. The total number and composition of neutrophils in the blood are therefore altered dramatically at this time.

    Fig. 1. Neutrophil consumption by tissues in the absence of bacterial infection.
    Neutrophils, primarily segmented neutrophils and to a lesser extent band neutrophils, migrate from the bone marrow into peripheral blood, before infiltrating organ tissues.

    It generally takes seven days for neutrophils to mature in the bone marrow, and the bone marrow pool contains neutrophils at various maturation stages, from immature myeloblasts, through promyelocytes, myelocytes, metamyelocytes, and non-segmented/band neutrophils, to segmented neutrophils, the most mature type of cell (Fig.1). During bacterial infection, the shortage of mature neutrophils in the peripheral blood means that more immature cells, such as myelocytes, metamyelocytes, and band neutrophils are also released this phenomenon is called ‘left shift’ (Fig. 2).

    The course of a bacterial infection can be divided into four phases using a combination of the white blood cell (WBC) count, which is almost the same as the neutrophil count, and the degree of left shift. During the first phase, occurring between 0 and 20 hours after the onset of infection, the neutrophil count, whether considering the left shift or not, briefly falls below the reference range. The consumption of neutrophils at the site of bacterial infection exceeds the capacity of the bone marrow to produce replacements. At the early stage, a left shift is not observed. During the second phase, which occurs between one and several days after the onset of infection, the neutrophil count increases as the supply from the bone marrow exceeds consumption at the infection site, and a left shift is observed. During the third phase, which occurs in the days following the second phase, high neutrophil counts continue to be seen, but without a left shift, as sufficient cells can be supplied to the infection site without increasing production in the bone marrow. By this stage, the bacterial infection is largely eliminated. During the fourth and final phase, occurring in the days following the third phase, the neutrophil count gradually decreases until it reaches the reference range, and no left shift is observed. At this stage the infection has been eliminated, and a large neutrophil population is no longer necessary.

    That said, some severe bacterial infections, including meningitis, infective endocarditis, and abscesses, may not show a left shift because the neutrophils in the blood are not continuously depleted, and so the production and release of immature neutrophils into the peripheral blood by the bone marrow is unnecessary.

    Fig. 2. High neutrophil consumption during bacterial infection.
    A large population of neutrophil cells, comprised of metamyelocytes and myelocytes in addition to segmented neutrophils and band neutrophils, migrate from the bone marrow into the peripheral blood before infiltrating the site of bacterial infection.

    Generally, there are dramatic changes in left shift and WBC count over the course of a bacterial infection, and a combination of these factors can be used for diagnostic purposes. Therefore, a time-series analysis of these parameters, comprising a minimum of two points, could improve the sensitivity and specificity of both the diagnosis of a bacterial infection and the evaluation of its progression. The left shift principally depends on the response of the bone marrow to neutrophil depletion from the blood, and so can be used to diagnose bacterial infections with high specificity. Therefore, if a left shift is observed upon admission, a bacterial infection should be suspected, and if this value increases within a few hours, a diagnosis of a bacterial infection can be made. Additionally, an assessment of both the left shift and WBC count can be used to evaluate the severity of bacterial infection and whether antibiotic treatment is appropriate.

    Takayuki Honda
    Department of Laboratory Medicine, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Japan